Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Eimeria tenella: Local Antibodies and Interactions with the Sporozoite Surface

.Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10³ oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10³ oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and imtnunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results. The immunofluorescence studies reveal the extremely labile nature of the sporozoite surface antigens and support the hypothesis that naturally elaborated IgA antibodies are involved in the modulation of complexed sporozoite surface antigens.     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

Eimeria tenella: local antibodies and interactions with the sporozoite surface

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

Changing patterns of serum and bile antibodies in re-infected rats with Clonorchis sinensis

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.     

Class IgG,IgM and IgA antibodies againstStaphylococcus aureus antigens in human serum and saliva

Using the ELISA method antibodies against the sonicate, teichoic acid (TA) and exoproducts ofStaphylococcus aureus were determined in sera and saliva of healthy individuals. Main serum antibodies against all the antigens used were shown to be class IgG antibodies. However, antigens of the sonicate stimulated significantly even the systemic IgA response. In the saliva class IgA antibodies predominated, but IgG antibody levels against TA and exoproducts approached the level of IgA antibodies. Levels of IgM antibodies against all antigens tested were low in both the serum and saliva which corresponds with the anamnestic type of response. On the basis of these results one may assume that not only IgG, but also IgA antibodies are important in the systemic immunity against staphylococcal infection and in the immunity of mucous membranes; besides IgA, even class IgG antibodies play an important role.     

Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals

To investigate the role of B cells and antibody in the immune response of mice to the murine intestinal parasite Giardia muris, we used mice treated from birth with rabbit anti-IgM antisera (aIgM). Such mice developed in serum and in gut secretions extreme Ig deficiency (IgM, IgA, and IgG) relative to control animals. The aIgM-treated mice showed no anti-G. muris antibody in serum or in gut wash material. Infections of G. muris in these mice were chronic, with a high load of parasite present in the small bowel, as reflected by prolonged cyst excretion (greater than 11 wk) and high trophozoite counts. In contrast, normal, untreated mice or NRS-treated animals developed anti-parasite IgA and IgG antibody in serum, demonstrated IgA antibody against the parasite in gut washings, and expelled the parasite within 9 wk. These effects of aIgM treatment on the murine response to primary infection with G. muris were demonstrated in two strains of mice: BALB/c and (C57BL/6 X C3H/He) F1. It was also observed that the response to G. muris infection in untreated animals was characterized by higher than normal total secretion of IgA into the gut and a concomitant increase in the serum polymeric IgA level. Mice treated with aIgM had a marked decrease of both monomeric and polymeric IgA in serum, and little detectable IgA in the intestinal lumen. These experiments provide the first demonstration that anti-IgM treatment suppresses a specific intestinal antibody response to antigen, and provide evidence that B cells and antibody play a role in the development of an effective response to a primary infection with G. muris in mice.     

The effect of host age on Rana temporaria-gorgoderina vitelliloba interactions

Mitchell J. B. 1982. The effect of host age on Rana temporaria-Gorgoderina vitelliloba interactions. International Journal for Parasitology12: 601–604. Two age groups of tadpoles, and newly metamorphosed and adult male Rana temporaria were fed the metacercarial cysts of Gorgoderina vitelliloba. In the younger tadpoles metacercariae died in their cysts. In the older tadpoles excystment took place and juvenile flukes invaded the kidneys, killing the hosts within 72 h. In newly metamorphosed frogs, an immunological response resulted in some of the juvenile flukes in the kidneys being attacked by eosinophils which adhered to and dissolved the tegument, presumably killing the flukes. In contrast, some young frogs were harmed by flukes in their kidneys. Migration away from the kidneys to the bladder took place on about the twelfth day after infection. Juvenile flukes in the kidneys of adult frogs 7 and 14 days after infection, evoked an inflammatory reaction involving polymorphs and lymphocytes. These cells did not appear to damage the parasites.     

Systemic and mucosal immune responses to sublingual or intramuscular human papilloma virus antigens in healthy female volunteers

The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19-31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12) or intramuscular injection (IM, n = 6). IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ~38-fold lower serum and ~2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans. TRIAL REGISTRATION: ClinicalTrials.govNCT00949572.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

Scanning electron microscopy of Fasciola hepatica L. during growth and maturation in the mouse.

Throughout the entire life of the fluke the spines anterior to the ventral sucker are arranged in approximately 60 rings each of 60 to 70 spines. The spines on the posterior body are scattered without any pattern of rings and by 1 week post infection (p.i.) their numbers have doubled (from 3,000 on the newly excysted juvenile) to 6,000 and by 3 weeks p.i. their numbers have multiplied by 8 to 24,000. Just prior to entry into the bile ducts, between 2 and 3 weeks p.i., all spines, anterior and posterior, have metamorphosed from single pointed to multipointed forms by division at the spine tips. Spines on the anterior body of mature flukes recovered from the bile ducts of mice 26 weeks p.i. have between 10 and 15 points whereas those on the posterior body have up to 30 points. The tegument forms a rectangular pattern of plateaux and valleys around each spine on the posterior body of mature flukes but this pattern is not present on the anterior body.     

Echinostoma caproni: kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice

The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels.     

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode

The surface antigens, which induce a serum antibody response during infection of mice with the intestinal trematode Echinostoma caproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDS-PAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000, 66,000, 75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacercariae of E. caproni.     

Trichinella spiralis: macrophage activity and antibody response in chronic murine infection

The role of macrophages, their products, and the specific antibody response were examined during chronic Trichinella spiralis infection in BALB/c mice. Adult T. spiralis in intestines were detected from 5 to 20 dpi. Muscle larvae numbers peaked at 45 dpi and thereafter a reduction was noted. The highest numbers of macrophages in the peritoneal cavity of infected mice were obtained up to 30 dpi. The production of NO by macrophages in infected mice was suppressed at 5 dpi, and then NO release increased until 45 dpi. The levels of NO in plasma and urine were lower in infected mice during the entire experiment in comparison to control. The production of O(2)(-) in peritoneal macrophages was inhibited during the first two weeks after infection and then increased until 90 dpi. Circulating T. spiralis antigens in plasma and urine were detected from 5 to 30 dpi. Specific IgM and IgA in serum increased until 20 dpi. IgG, IgG(1), and IgG(2) levels in serum increased until 60 dpi.     

The localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.     

IgA allotype suppression in mice: A cellular implication for the IgA preponderance in the gut

Paternal IgA allotype suppressions were induced in (A/HeJ x SJL/J)F₁, (BALB/cJ x CB-20) F₁, and (BALB/cJ x SJL/J)F₁ mice by perinatal exposure of the neonates to antibody against paternal IgA allotype (Ig-2b). All the Ig-2b allotype suppressions were temporary and were prominently seen at the age of 4–10 weeks. These transient Ig-2b allotype suppressions were manifested by a suppression of Ig-2b allotype in the serum and a suppression of Ig-2b allotype-producing cells in the gut lamina propria. During the suppression, no replacement of the Ig-2b allotype by IgM and IgG plasma cells was seen in the gut lamina propria. The transient IgA allotype suppression was primarily due to a depletion of IgA allotype precursors in Peyer's patches; suppressor T cells were not actively involved. These results suggest that IgA preponderance in the gut is due to the migration of the patch IgA precursors and their accumulation and maturation in the gut lamina propria.