Lagochilascaris minor: antibody production in experimentally infected mice / Lagochilascaris minor: produção de anticorpos em camundongos experimentalmente infectados

Lagochilascaris minor is the causative agent of lagochilascariosis, a disease that affects the neck region and causes festering abscesses, with eggs, adult parasites and L3/L4 larvae within the purulent exudates. Today, mice are considered to be intermediate hosts for the parasite. C57BL/6 mice produce immunoglobulin IgM, IgA and IgG against the crude extract of the parasite; on the other hand, antibodies produced against the secreted/excreted antigens of Lagochilascaris minor present lower levels of IgM, IgA and IgG. This is the first description of antibody detection against different antigens of Lagochilascaris minor.

Lagochilascaris minor é o agente etiológico da lagochilascariose, uma doença que afeta a região do pescoço causando abscessos exudativos com presença de ovos, parasitos adultos e larvas nos de exudatos purulentos. Hoje em dia, camundongos são considerados os hospedeiros intermediários para o parasita. Camundongos C57BL/6 produziram imunoglobulinas IgM, IgA e IgG contra o extrato bruto do parasita; por outro lado, anticorpos produzidos contra os antígenos secretados/excretados de Lagochilascaris minor apresentaram níveis mais baixos de IgM, IgA e IgG. Esta é a primeira descrição da detecção de anticorpos contra diferentes antígenos de Lagochilascaris minor.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

An antigen detection assay for diagnosing filariasis.

In this study we examined the diagnostic potential of monoclonal antibodies (MAb) reactive to antigens of adult Brugia malayi, their microfilariae and antigen of Dirofilaria immitis. The MAb of clone 17E10, which were of IgM isotype, reacted to the inner cuticles and internal content of both male and female worms and also to the sheath and internal content of microfilariae in utero. However, these MAb did not react to the sheath of blood circulating microfilariae. The MAb 17E10 produced a smear pattern between 37 to > 200 kDa in the Western blot analysis against a SDS-PAGE separated extract of B. malayi. The epitopes were non-protein in nature as indicated by their resistance to proteinase-K treatment. The MAb 17E10 were applied in a sandwich ELISA to detect filarial antigen in the buffy coat and plasma of patients. We tested patients with different clinical manifestations of brugian filariasis, i.e. microfilaremia (M), lymphangitis (L) and elephantiasis (E), as well as non-symptomatic inhabitants of a filariasis endemic area (NE), and compared them to samples from non-symptomatic inhabitants of disease non-endemic areas (NNE). It was found that 22 of 31 (70.9%) of M, 7 of 13 (53.8%) of L, 2 of 14 (14.2%) of E, 10 of 100 (10.0%) of NE and none (0%) of the NNE were positive for antigenaemia. The assay was also positive in 14 of 15 (93.3%) blood samples from B. malayi microfilaremic cats and in 7 of 7 (100%) blood samples of Dirofilaria immitis microfilaremic dogs. The so-developed test has a high potential for routine diagnosis of active filariasis, for epidemiological studies in both humans and reservoir animals and for monitoring treatment efficacy.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses

Studies on the development of DNA vaccine against Cysticercus cellulosae infection and its efficacy.

DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase.

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

Kinetics of humoral & cellular immune responses in experimental cysticercosis in pigs infected with Taenia solium.

Studies were undertaken to assess the kinetics of antibody responses, lymphocyte transformation to Taenia solium larval antigens (crude soluble extract antigen and antigen B), and T cell subpopulation in piglets following experimental infection. Cysticercosis was established in 1-2 month old piglets after feeding 5,00,000 T. solium eggs per pig. The anti-CD4 and anti-CD8 monoclonal antibodies against swine T cells were raised indigenously. It was observed that at 60 days post infection (PI) there was a significant increase (P < 0.01) in CD4+ T cells without any change in CD8+ T cells. Increased 3H-thymidine uptake was found in infected piglets at 45 days PI using both CSE and antigen B. Kinetics of antibody responses indicated significant increase (P < 0.01) at 15 days PI (with CSE antigen) and 30 days PI (with antigen B) by ELISA. This increase persisted till 90 days PI (the time up to which the animals were followed). It was also observed that the cellular mechanisms were triggered in late stage (60 days PI) as compared to humoral responses (15-30 days PI) and may persist longer as seen by both lymphocyte transformation and T cell subpopulation studies. The study suggests that in cysticercosis, both humoral and cellular mechanisms may play a role in the host defences.

Fasciola gigantica: studies of the tegument as a basis for the developments of immunodiagnosis and vaccine.

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.

Correlation between degree of protection and humoral antibody response in hamsters immunized with somatic and excretory secretory antigens of Ancylostoma ceylanicum.

Correlation between the degree of protection and induced serum antibody response in hamsters immunized with somatic and excretory-secretory (ES) antigens of adult. A. ceylanicum was investigated. Hamsters were immunized with non purified and Sephadex G-200 fractions (F1, F2 and F3) of somatic and ES antigens. The degree of protection was assessed in terms of percent worm reduction compared to controls against challenge infection. Induced humoral antibody response was determined by ELISA. Both somatic and ES antigens had shown moderate to significant protection but the latter was found more immunogenic as highest level of protection (67.02%) was achieved by these antigens. Humoral antibody was found highly elevated in animals immunized with protective doses of somatic and ES antigens. The maximum serum antibody titer i.e. 1:3200 was noticed in animals immunized with high protective dose (64.59% protection) of fraction F1 of ES antigens. Antibody titer correspond to the degree of protection and a positive correlation between induced humoral antibodies and protection level was established.

Evaluación de la prueba de Dig-Elisa para la detección de anticuerpos anti-fasciola hepática en ganado bovino / Evaluation of Dig-Elisa test for detection of anti-fasciola hepatica antibodies in bovine cattle

Se implementó la prueba de Dig-Elisa para detectar IgG anti-F. hepática en bovinos, estableciendo la sensibilidad y el valor de corte de ésta. Los promedios de diámetros de la zona de reacción (DZR) ñ desviación estándar en mm, determinados con Dig-Elisa en 2 grupos de sueros de bovinos: negativo (n=335) fueron 2,5 ñ 2,6 y 12,5 ñ 2,2, respectivamente; obteniéndose una sensibilidad de 98,5 por ciento, una especificidad del 100 por ciento y valores predictivos positivo y negativo de 100 por ciento y 91 por ciento, respectivamente, al utilizar un valor de corte de 8 mm. Adicionalmente, se analizaron las variaciones mensuales, durante un año, de IgG anti-F. hepática y de huevos por gramo (hpg) del parásito eliminados en las heces de un hato de bovinos Holstein hembras en producción (n=30), de Tulancingo (zona enzoótica de fasciolosis), Estado de Hidalgo, México. Los DZR obtenidos con los sueros de los bovinos de Tulancingo nunca bajaron de 11 mm, observándose valores máximos en los meses de septiembre de 1991 (15,6 ñ 2,7) y junio de 1992 (14,6 ñ 1,1). Los valores mínimos y máximos de hpg de heces se obtuvieron en septiembre (0,09) y en mayo (28,5) respectivamente. Los valores obtenidos con Dig-Elisa tuvieron menor variación que los del examen coprológico. Se concluye que el Dig-Elisa puede ser de utilidad en estudios epidemiológicos de fasciolosis bovina

Antibody response in schistosomiasis mansoni: IgG and IgE response to three anti-schistosomal candidate vaccines glutathione -S- transferase, schistosoma mansoni 217 and schistosoma mansoni 23

A total of 199 serum samples from individuals living in a Schistosoma mansoni endemic Egyptian village were tested for their IgG and IgE response to various schistosomal antigens using ELISA. In addition, it was possible to examine the IgE response in serum samples from 54 S. mansoni infected and uninfected Sudanese subjects. IgG responses were examined against AWA, SEA, Sm GST, Sm 21.7, Sj 26, KLH and Sm 23. IgE responses were examined to these antigens as well as to a detergent extract of adult worm antigen [DAWA] and a schistosomular antigen [Somule Ag]. In the Egyptian patients studied, there was a significant negative correlation between IgG response to Sm GST intensity of infection. In male patients there was a significant difference between negative and positive IgG responses to AWA and Sm 21.7 in relation to the intensity of infection with the positive responses being associated with lower intensities of infection. However, there was no relationship between age and different IgG or IgE responses. The infected and uninfected persons did not differ significantly in their responses. In contrast, most of the IgE responses in Sudanese patients were significantly higher than those of the uninfected persons. Moreover, there was a significant negative correlation between age and IgE responses to KLH and Sm 23. Despite the contrast between the Egyptian and the Sudanese responses, the study shows an interesting pattern of antibody response to three candidate vaccines [Sm GST, Sm 21.7 and Sm 23]

Comparison of the protective efficacy on mekongi schistosomiasis in mice induced by antigens derived from cercariae, schistosomulae and adult worms.

Protective efficacy of the extracts of cercariae, schistosomulae and adult worms of S. mekongi was studied in mice receiving immunizations with these extracts emulsified with Freund's complete adjuvant initially and incomplete adjuvant subsequently, and compared with mice receiving physiological saline with or without adjuvants as controls. After challenge with cercariae, the animals were sacrificed and the larvae or adult worms harvested by lung recovery and perfusion techniques on day 5 and weeks 6-8, respectively. Worm reduction rates were significantly higher in mice receiving extracts of schistosomula (59%) and adult worms (51%) than in those receiving the cercarial extracts (31%). Similar findings were obtained with the perfusion technique showing worm reduction rates of 57%, 53% and 30% in mice receiving extracts of schistosomulae, adult worms and cercariae, respectively. ELISA antibody titers were correspondingly increased in mice receiving extracts of schistosomulae and adult worms, but not in those receiving cercariae. This apparent association may be inadequate to suggest that the increase in ELISA titer be used as an indicator for resistance in mekongi schistosomiasis.

Course of antibody production by the DIG-ELISA method in neonatally infected and juvenile infected rats after primary infection with Breinlia booliati (Filarioidea: Onchocercidae).

The course of antibody production in Wistar neonatal and juvenile rats after primary infection with Breinlia booliati was studied by the DIG-ELISA technique using filter papers impregnated with capillary blood drawn from the infected rat tails at 7, 14, 28, 60 and 90 days post infection. Sera of neonatally infected rats did not react with adult worm antigen until day 7 and the titers of antibody remained at very low levels for the next 7 days. There was little tendency to eliminate the filarial larvae during this time. The antibody levels then rose rapidly throughout the next fortnight and increased to a maximum at day 60 after which the titer leveled out at a constant high value until early patency at day 90. On the other hand, antibodies could be detected in sera of juvenile infected rats as early as day 7 and the levels of antibody rose markedly to a maximum at day 28. During the period from day 60 to day 90 at early patency, the antibodies declined gradually to lower levels. The humoral immune responses of 42 neonatally infected rats and 53 juvenile infected rats of 3 strains (Lewis, Wistar and Sprague Dawley) were tested against soluble B. booliati antigens from both female (1:50) and male (1:10) worm extracts by the DIG-ELISA method. Antibodies were detected in sera from all the microfilaremic and amicrofilaremic rats belonging to neonatally and juvenile infected groups. Sera of clean neonatal rats did not give a positive reaction zone.

Evaluation of RNA rich fraction of Litmosoides carinii in induction of protection in infected rats.

The RNA rich fraction of adult L. carinii worms was evaluated in evoking a protective response in infected rats. The RNA immunization was seen to be effective in limiting the microfilaraemia in peripheral blood as well as the adult worm burden. The antibodies to both RNA antigen and adult worm antigen were high in this group of animals at the peak of infection. The RNA immunization was seen to evoke hyperresponsiveness in lymphocytes to mitogens like adult worm antigen, PHA and Con A.

Suppression of the IgE antibody response by Ascaris suum components: effect of x-irradiation

The effect of X-irradiation on the supperession of IgE antibody responses induced by some of the Ascaris suum (ASC) components was analyzed in mice (7-week old A?Sn females). Treatment with 300 R 24h before immunization with 50 *g OVA and 200 *g ASC suppressive components abolished the damping effect on ati-OVA IgE antibody levels. The same effect was observed on the anti-ASC IgE antibody response obtained in mice injected with 200 *g ASC immunogenic plus 200 *g ASC suppressive components. Moreover, the failure of suppressive components to induce an IGE anti-ASC antibody response on their own was also abolished by X-irradiation. These results indicate that the suppressive components are able to elicit an IgE antibody response, but simultaneously activate a regulatory mechanism which suppressive both the homologous (anti-ASC) and heterologous (anti-OVA) antibody formation

Iodinated soluble adult worm protein preparations of Schistosoma mansoni do not induce immediate hypersensitivity reactions but retain other immunogenic properties

Samples of Schistosoma mansoni soluble adult worm proteins (SWAP) were iodinated with 4-15 µmolI/mg protein using iodine monochloride. The capacity to elicit immediate hypersensitivity reactions of the iodinated derivatives was compared to that of the native SWAP preparations. The degranulation of mast clls from infected mice decreased with increasing iodine incorporation and was absent in fully iodinated samples containing 15 µmol I/mg protein. The response of guinea pigs and humans to the intradermal test with iodinated SWAP also decrease in proportion to iodine incorporation, and no responses were obtained with fully iodinated samples. No false-positive tests were observed. Antibodies to the folly iodinated extracts generated in C57BL/10 mice reacted by ELISA with the unmodified proteins and by immunoprecipitation on agar gel. The immunoprecicpitation pattern suggested that some epitopes were altered by iodination

Further characterization of ascaris suum component(s) with supressive activity on the IgE antibody response

In order to characterize the component(s) of Ascaris suum reponsible for damping of the IgE antibody production we demonstrated that the extract incubated in sodium acetate buffer, pH 4.5, maintained its suppressive effect and the same protein banding pattern by SDS-PAGE. Elimination of the lipoprotein components of the extract also left its damping properties unchanged. SDS-PAGE of the lipoprotein-free extract revealed practically the same pattern as shown by the whole extract, except for the high molecular weight polupeptides. These results indicate that the suppressive component(s) of A. suum did not precipitate and retained their activity at low pH. In addition, they appear not to be lipoproteins

Demonstration of species-specific antigens of Gnathostoma spinigerum, a preliminary attempt.

An attempt was made to demonstrate the presence of species-specific antigens for Gnathostoma spinigerum advanced third-stage larvae (GsAL3) in a rabbit receiving weekly immunization with GsAL3 for seven weeks. The homologous and heterologous antibodies against GsAL3 and G. doloresi adult worm (Gd) antigens were initially detected by immunoelectrophoresis (IEP) and ELISA after the second immunization, and their levels were gradually increased with the number of immunizations. Though cross-reactivity between GsAL3 and Gd were shown with both tests, species-specific antibodies for the homologous antigens were demonstrated. After cross-absorption of rabbit hyperimmune serum was collected after the seventh immunization, seven 'putative' species-specific precipitin bands of GsAL3 were identified. The ELISA values of the rabbit hyperimmune serum showed 50% inhibition after absorption with 0.7 micrograms/ml of homologous GsAL3 antigens as opposed to 1.0 micrograms/ml of the heterologous Gd antigens.