A comparison of selected immunological techniques used to detect anti-leishmanial antibodies in the sera of two reptile species.

European green lizards (Lacerta viridis) and spiny-tailed agamids (Agama caudospinosum) were obtained from areas endemic for human leishmaniasis. Serum antibody titres against Leishmania agamae, a reptilian leishmanial species, in normal lizards and lizards injected with Leishmania agamae promastigotes were measured by 5 immunological methods commonly used in the serodiagnosis of the human and mammalian leishmaniasis viz. immobilisation test (IMM), direct agglutination (DA), complement-fixation test (CFT), indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA). Correlation coefficients (r) were determined for comparisons between each method and linear regression equations calculated to convert antibody titres by one method to those by another. In each lizard species, the IMM test gave the lowest values while the highest were obtained with ELISA. The highest mean titre obtained by ELISA was between 2 and 10 times that obtained by the other methods for both control and immune sera. The methods of preparing the leishmanial antigen extracts affected the IHA and ELISA titres, while the source of complement was critical in obtaining good CFT values. Correlations ranging from 3% to 77% were found for the control animals but higher values ranging from 65% to 96% were obtained with the immunised lizards. Overall, the best correlation was with IHA and ELISA (r greater than 0.82) and with ELISA values for different antigen preparations compared with each other for both control (r greater than 0.67) and immune (r greater than 0.90) sera. ELISA thus appears the most sensitive method for detection and quantitation of anti-flagellate antibodies in normal lizard serum and for the determination of titres in immune serum. ELISA is the most applicable technique for screening reptiles and other lower vertebrates for anti-parasite immunoglobulins, and for screening potential carriers or reservoirs of infective flagellates in epidemiological studies aimed at disease control, especially in areas where human infections are prevalent.

Lectins and lectin-like molecules in lower plants. II. Freshwater algae.

Freshwater chlamydomonad algae possess flagella surface isoagglutinins of a glycoprotein nature which are involved in gamete recognition and consequent cell fusion. Alternatively, such molecules may have a receptor role in certain Chlamydomonas species. In some freshwater volvocaines, lectin-like substances act as inducers of sexuality. At the moment it is speculative whether these materials can serve as naturally-occurring antibodies to protect algae from environmental antigens.

Lectins and lectin-like molecules in lower plants. I. Marine algae (review).

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.

Antigen distribution and humoral response in the lizard, Agama caudospinosum, after injection with Leishmania agamae.

The response of the spiny-tailed agamid lizard, Agama caudospinosum, to administration of Leishmania agamae promastigotes was investigated. Lizards given a single injection of promastigotes showed no signs of clinical infection. Neither promastigotes nor amastigotes were found in blood and tissue impression smears, nor in blood and selected body organ cultures. However, parasite antigens were demonstrated by an immunoenzyme method only in the liver, small intestine, stomach, spleen and kidney. Non-precipitating serum antibodies with gamma-electrophoretic mobility were detected by enzyme-lined immunosorbent assay 1 week post-injection and a maximum titre was reached after 6 weeks. The mean immune serum protein concentration increased significantly (P less than 0.005) about two-fold over the controls after injection. Decreases occurred in the beta-globulin region of anti-L. agamae sera (P less than 0.01) whilst the gamma-globulin fraction was increased (P less than 0.005) following electrophoresis on cellulose acetate membranes. C-reactive protein was not detected in any of the sera. These data show that although A. caudospinosum failed to become infected by L. agamae promastigotes, which had been isolated from agamids, it did exhibit antigen distribution and a humoral response similar to other reptiles.

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.

The effect of dose, route, number of injections and the use of adjuvant on the clearance of and immune response to, haemocyanin following injection into brown trout (Salmo trutta).

The primary and secondary immune responses were investigated in trout injected with haemocyanin (HCN) intramuscularly (IM) or intraperitoneally (IP), with or without adjuvant. HCN was detected in the blood 30 min after injection and clearance times varied after one injection from 8 to 56 days. Fish given two and three injections cleared the antigen faster. Precipitins against HCN were first detected 21 to 30 days after injection and were still present on Day 56. However, antibodies detectable by indirect haemagglutination (IHA) and complement fixation (CFA) were initially demonstrated 7 to 21 days after a single injection and highest titres were reached between 33 and 56 days according to the experimental protocol. In fish given two injections, maximum titres were reached between Days 42 and 56 (IM), and Days 50 and 56 (IP). With three injections, maximum CFA and IHA values occurred on Days 62 and 66 respectively in the case of IP, and on Days 103 and 106 respectively with IM. Overall, higher titres were found with IHA than by CFA.

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.

Trypanosoma brucei products activate components of the reactive response in astrocytes in vitro.

A study was made of the effects of T. b. brucei and the disrupted parasite material on different components of the reactive response of astrocytes in primary cultures prepared from neonatal rats and C6 glioma cells. The effects were compared with lipopolysaccharide (LPS) and fragments of the C6 cells. The disrupted trypanosome material, LPS and C6 fragments caused dose--and time--dependent alterations in morphology of the primary cultures from flat to stellate shape, increases in levels of glial fibrillary acidic protein (GFAp) and enhanced MHC class I and II expression. Exposure to trypanosome material and C6 fragments caused marked increases in phagolysosomes and lysosomes in the primary cultures. The findings demonstrate that T. b. brucei products and astrocyte cell debris are internalized and initiate astrogliosis in vitro.

The immune response of brown trout (Salmo trutta L.) to injection with soluble antigens.

The immune response of brown trout (Salmo trutta) to horse serum and keyhole limpet haemocyanin was studied. Intraperitoneal and intramuscular injections were used, with and without adjuvant, in 209 fish. Complement-fixing antibodies (CFA) and precipitins were produced to both antigens. CFA were detected after 8 days to haemocyanin and after 13 days to horse serum. Maximum CFA titres to a single intraperitoneal injection of horse serum or haemocyanin were reached at 44 and 43-46 days respectively. Precipitins to a single injection of haemocyanin given intraperitoneally were detected after 19 days using gel diffusion. Similarly using the intramuscular route they were detected after 22 days. However, using counter-current electrophoresis, precipitins were detected after 8 days by the intraperitoneal route and after 9 days by the intramuscular. Precipitins to horse serum given intraperitoneally were demonstrated after 22 days by both gel diffusion and counter-current electrophoresis. Fish given 2 intraperitoneal injections of haemocyanin in adjuvant reached maximum CFA titres after 55 days; a 3rd injection on day 56 did not produce a marked increase in titre. Fish given intramuscular injections of haemocyanin in adjuvant showed maximum CFA titres at day 43. After a 3rd injection on day 56, maximum CFA titres were reached between days 92 and 106. Intramuscular injections gave significantly higher titres than those given by the intraperitoneal route. Some fish which showed no precipitins by gel diffusion were positive by counter-current electrophoresis. Precipitating antibodies to haemocyanin migrating in the beta2-gamma1 region were detected by immuno-electrophoresis.

Salivary gland surface carbohydrate variations in three species of the Anopheles gambiae complex.

Fluorescein isothiocyanate (FITC)-conjugated lectins (agglutinins) were employed as probes to distinguish between the various carbohydrates present on the surface of salivary glands of three species of mosquito of the Anopheles gambiae complex. Of twenty lectins tested, eight (Concanavalin A- Con A, Lathyrus odoratus- LOA, Lens culinaris, Pisum sativum-PSA, Vicia faba- VFA, Triticum vulgaris, Maclura pomifera- MPA and Ulex europaeus) specifically reacted with the salivary gland membrane. Both mannosyl and N-acetylglucosamine moieties were detected in the three Anopheles gambiae sensu stricto strains, the two An. arabiensis strains and a single An. merus strain examined. Variations in the degrees of fluorescent intensities of Con A and MPA in particular suggested interspecies differences in membrane mannosyl and galactosyl residues on the salivary gland lobes of the three mosquito species in this study. Furthermore, intraspecific variations in mannose as indicated by Con A, LOA, PSA and VFA staining were demonstrated between the An. gambiae s.s. strains. The use of either peroxidase-labelled or biotinylated lectins confirmed the binding specificities of the above lectins. The consistent differences observed in lectin binding suggest that variations occur in salivary gland surface carbohydrate residues and that lectins can be used to distinguish between at least some members of the An. gambiae complex.

The primary immune response of brown trout (Salmo trutta) to cellular and soluble antigens: Enumeration of antibody-secreting and antigen-binding cells, and the production of antibody.

Significantly increased numbers (P less than 0.01) of antigen-binding (ABC) and antibody-secreting cells (ASC) were stimulated, in the spleen and the anterior kidney (AK) of the brown trout (Salmo trutta), by a single intraperitoneal injection of Salmonella typhimurium lipopolysaccharide (LPS), keyhole limpet haemocyanin (KLH), sheep red blood cells (SRBC) or KLH- or LPS-coated SRBC. With the exception of the SRBC group, the spleens contained more AVC and ASC per 10(6) lymphoid cells than the AK. Fewer ASC were found when guinea pig complement was used than with salmon serum; with 3 exceptions the differences were significant. In both organs, ABC and ASC were detected between 2 and 4 days after injection and maxima were reached between day 14 and day 18 in most of the experimental groups. However, with fish immunised with SRBC, ABC and ASC first occurred on day 6 and peak counts were obtained on day 12. In all groups the counts had returned to background levels by day 28. The coupling of LPS or KLH tro SRBC, with one exception, significantly increased the numbers of ABC and ASC (p less than 0.02) compared to when uncoated SRBC, LPS or KLH were injected. There was no significant change when the ABC results for antigen-coated SRBC were compared to those obtained when LPS was injected by itself. Following antigenic stimulation there were significant increases (p less than 0.05) in most cases in both the spleen and the AK to body weight ratios. There were significant differences in both the spleen and the AK to bodyweight ratios. There were significant differences (p less than 0.001) between KLH and KLH-SRBC (spleen and AK), LPS and LPS-SRBC (spleen and AK) and SRBC and LPS-SRBC (AK alone). Antibodies were detected 2 to 8 days after sensitised cells were first found and reached maxima 5 to 16 days after the cellular responses. The titres obtained with experimental sera were significantly different (p less than 0.001) compared to the appropriate control groups.

Effects of physico-chemical treatments on haemagglutination activity of Anopheles gambiae haemolymph and midgut extract.

Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature--not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile > 40 degrees C, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithiothreitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca2+ ion, albeit to a lesser extent Mg2+ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M(r)) of 65 kDa whilst that of haemolymph was 40 kDa. This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M(r) of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).

The primary humoral immune response of European green lizards (Lacerta viridis) to Leishmania agamae.

European green lizards, Lacerta viridis, produced relatively thermostable, dithiothreitol-sensitive, non-precipitating, agglutinins and complement-fixing antibodies (CFA) to Leishmania agamae administered subcutaneously (SC), intraperitoneally (IP) or orally (OR). Antibodies were also detected by the immobilization test (IMM) and by enzyme-linked immunosorbent assay (ELISA). The most sensitive method for the detection of stimulated immunoglobulins was ELISA. Antibodies were detected as early as 3 days post-infection with ELISA and between 5 and 7 for CFA, direct agglutination (DA) and indirect haemagglutination (IHA). In the case of IMM, the times of first detection varied from 14 to 28 days. Maximum CFA (2(-8)), DA (2(-8)), IHA (2(-11)) and ELISA (2(-16)) titres were reached from 42 to 49 days with significantly higher values occurring in the OR and IP groups. With IMM, maxima occurred after 5 or 6 weeks. Following exposure, two- to five-fold significant increases in serum lysozyme levels were demonstrated but the concentrations in sera following SC, IP or OR routes of antigen administration were not significantly different when the groups were compared with each other. The highest lysozyme values (approximately 12.3 - 12.5 micrograms ml(-1)) were found in the SC and OR groups when compared to the IP (7.40 micrograms ml(-1)).

The primary immune response of the green toad (Bufo viridis) to challenge with Crithidia fasciculata.

The primary immune response of the green toad (Bufo viridis) following immunization with Crithidia fasciculata choanomastigotes was studied. Lysins, agglutinins, and antibodies detectable by enzyme-linked immunosorbent assay (ELISA) were first detected in the sera of immunized animals one week after injection. The antibody titers increased to significant levels (P less than 0.01) and maximum values were reached seven weeks post-immunization. The stimulated immunoglobulins were antigen-specific, partially heat-labile, sensitive to the reducing agent dithiothreitol, possessed precipitin activity, effectively fixed complement and exhibited an electrophoretic mobility similar to the gamma-globulins of human serum. On this basis, it is probable that the antibody produced during the primary response in green toads is high molecular weight IgM. Increases in serum lysozyme levels paralleled the rise of antibody titers. Overall, the lysozyme concentration increased two-fold compared to the appropriate controls. This is the first report of the immune response in amphibians to experimental injection with protozoan parasites and the use of the ELISA technique to detect antibodies in amphibian sera.

Haemagglutinins and parasite agglutinins in haemolymph and gut of Glossina.

Agglutinating activity was found in the haemolymph and in extracts of midgut and hindgut of Glossina austeni against calf, guinea pig and chicken erythrocytes for the first time. Trypanosoma brucei procyclic forms were also agglutinated by midgut and hindgut extracts, but not by haemolymph. The 3 fractions tested failed to react with erythrocytes of four other vertebrates and other trypanosomatids (Leishmania hertigi and Crithidia fasciculata) examined. The reciprocal titre of agglutinating activity of the 3 fractions against calf, guinea pig and chicken erythrocytes were approximately the same at a dilution of 64. Both midgut and hindgut agglutinins showed higher titres against T. brucei at reciprocal dilutions of 128 and 256 respectively. Sialidase treatment, reduced the agglutinating activity to half its normal range while trypsinisation had no effect. The results of attempts to inhibit the agglutinating activity using a variety of sugars and glycoproteins revealed that midgut and hindgut were specific for D+glucosamine suggesting that midgut and hindgut agglutinin were possibly of the same origin. This is the first report of haemagglutinating and parasite agglutinating activity in Glossina tissues which may play a role not only in insect defence reactions but also in the determination of parasite establishment and infection in vectors.

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

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. Titres were highest in the haemolymph and midgut extracts versus human AB, horse, chicken and goat RBCs and in hindgut against human AB, chicken and sheep; foregut extract gave relatively low titres. Adsorption of haemolymph and gut extracts with selected RBCs coupled with carbohydrate inhibition and the use of enzyme-treated RBCs revealed the presence of multiple (hetero-) agglutinins. An.gambiae lectins were specific for (1-1)-, (1-4)- or (1-6)-linked glucose based disaccharides, glucose and its (1-2) or (1-3) linkages with fructose and, to a lesser extent, aminated or N-acetylated glucose, or galactose and its deoxy derivatives. This study presents the first report of the occurrence of heterogenous anti-RBC agglutinins in haemolymph and gut extracts of the mosquito An.gambiae, together with the sugar-binding specificities of these lectins.

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 agglutination of erythrocytes and Leishmania parasites by sandfly gut extracts: evidence for lectin activity.

Lysates of heads, hind- and midguts of male and female Phlebotomus papatasi were found to contain lectins or lectin-like molecules capable of agglutinating human red blood cell types of the ABO(H) system and promastigotes of Leishmania aethiopica, L. major and L. donovani but not L. hertigi hertigi promastigotes or Crithidia fasciculata choanomastigotes. The agglutination of erythrocytes from the human O Rhesus positive blood group by sandfly midgut extracts was inhibited by two disaccharides; trehalose and turanose. This is the first report of haemagglutinating and parasite agglutinating activity in sandflies (Psychodidae).

Mast cells, histamine and the pathogenesis of intestinal damage in experimental Trypanosoma brucei brucei infections.

Intestinal damage with increased permeability is a prominent feature of experimental African trypanosomiasis. The possible involvement of mast cells and histamine in the altered gut integrity was investigated, at the level of the jejunum, in BALB/c mice infected with Trypanosoma brucei brucei. Mast cells were studied by selective staining of granule content with Alcian Blue/Safranin and quantitative histology, and histamine concentrations were determined by a fluorimetric method. Mast-cell activation, shown by a marked reduction in the numbers of positive-staining cells seen per villous section, was prominent on days 7 and 14 post-infection (there was, for example, a reduction to 36% of the control value by day 14; P=0.0001). By day 21, however, there were 131% more staining cells per villous section in the infected mice than in the uninfected controls (P=0.003). Histamine levels in homogenates of the jejunal mucosae of the infected mice were found to be significantly elevated at each time-point. The maximum increase was observed on day 14, when the numbers of granulated mast cells were at their lowest, with mean (S.E.) concentrations of 6.744 (0.890) ng/mg tissue for the infected mice and 2.813 (0.321) ng/mg for the uninfected controls (P=0.0008). The jejunal mucosa suffered progressive morphological damage during the infection, with oedema of the lamina propria and villi and disruption of the endothelium. These results indicate that mast cells are involved with the intestinal pathology that develops during experimental African trypanosomiasis.