Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.

The effects of nitric oxide on the immune response during giardiasis

Nitric oxide (NO) is a free radical synthesized from L-arginine by different isoforms NO-synthases. NO possesses multiple and complex biological functions. NO is an important mediator of homeostasis, and changes in its generation or actions can contribute or not to pathological states. The knowledge of effects of NO has been not only important to our understanding of immune response, but also to new tools for research and treatment of various diseases. Knowing the importance of NO as inflammatory mediator in diverse infectious diseases, we decided to develop a revision that shows the participation/effect of this mediator in immune response induced against Giardia spp. Several studies already demonstrated the participation of NO with microbicidal and microbiostatic activity in giardiasis. On the other hand, some works report that Giardia spp. inhibit NO production by consuming the intermediate metabolite arginine. In fact, studies in vitro showed that G. lamblia infection of human intestinal epithelial cells had reduced NO production. This occurs due to limited offer of the crucial substrate arginine (essential aminoacid for NO production), consequently reducing NO production. Therefore, the balance between giardial arginine consumption and epithelial NO production could contribute to the variability of the duration and severity of infections by this ubiquitous parasite.

Avaliação da dosagem de interleucina-5 e imunoglobulina e em pacientes com giardíase com ou sem eosinofilia / Evaluation of dosage of interleucina-5 and immunoglobulin e in patients with giardíase with or without eosinophilia

Giardíase é uma protozoose intestinal causada por Giardia lamblia. A eosinofilia (>500/mm3) nas parasitoses é geralmente encontrada nas infecções por nematódeos e raramente é referida na literatura em relação à infecção por G. lamblia. Este estudo foi desenhado a fim de determinar a relação entre IL-5 e presença de eosinofilia em crianças e adolescentes com giardíase. Foram selecionadas 38 crianças e adolescentes na faixa etária de 0-18 anos portando exclusivamente G. lamblia e 32 crianças e adolescentes com giardíase. Foram selecionadas 38 crianças e adolescentes. Foram excluídas nesta fase do projeto pacientes portadoras de outros parasitas. No momento da entrega do material fecal era coletado sangue para a realização do hemograma, dosagem da IgE e IL-5. A casuística foi dividida em 3 grupos. Grupo 1: portadores de giardíase e eosinofilia (N:20), Grupo 2: portadores de giardíase sem eosinofilia (N:18) e Grupo 3: pacientes com ausência de parasitose (N:32). Resultados: Nos casos de giardíase, 52.6 apresentaram eosinofilia. Grupo 1: 100.

Giardia y giardiasis / Giardia and giardiasis

Giardia lamblia es un protozoario parásito que habita el intestino delgado de los seres humanos y de muchos otros vertebrados y es una de las más comunes causas de diarrea en todo el mundo. Durante su ciclo de vida Giardia sufre significativos cambios bioquímicos y morfológicos que le permiten sobrevivir en ambientes y condiciones que de otro modo lo destruirían. Para sobrevivir fuera del intestino del hospedador, los trofozoítos de Giardia se diferencian a quistes, los que se caracterizan por poseer una rígida pared glicoproteica externa que les permiten sobrevivir inclusive frente a la acción de los desinfectantes más comunes. Otro de los mecanismos de adaptación de este parásito es la variación de los antígenos de superficie que le permite a los trofozoítos evadir la respuesta inmune del huésped y generar infecciones tanto agudas como crónicas o recurrentes en individuos infectados. Durante los últimos años se han producido importantes avances en el conocimiento de las bases moleculares de los procesos de enquistamiento y variación antigénica en Giardia que pronostican el pronto hallazgo de nuevos agentes quimioterapéuticos y/o inmunoprofilácticos contra este importante parásito intestinal.

Immunopathology of giardiasis: the role of lymphocytes in intestinal epithelial injury and malfunction

T lymphocyte-mediated pathogenesis is common to a variety of enteropathies, including giardiasis, cryptosporidiosis, bacterial enteritis, celiac's disease, food anaphylaxis, and Crohn's disease. In giardiasis as well as in these other disorders, a diffuse loss of microvillous brush border, combined or not with villus atrophy, is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water, which ultimately cause diarrheal symptoms. Other mucosal changes may include crypt hyperplasia and increased infiltration of intra-epithelial lymphocytes. Recent studies using models of giardiasis have shed new light on the immune regulation of these abnormalities. Indeed, experiments using an athymic mouse model of infection have found that these epithelial injuries were T cell-dependent. Findings from further research indicate that that the loss of brush border surface area, reduced disaccharidase activities, and increase crypt-villus ratios are mediated by CD8+ T cells, whereas both CD8+ and CD4+ small mesenteric lymph node T cells regulate the influx of intra-epithelial lymphocytes. Future investigations need to characterize the CD8+ T cell signaling cascades that ultimately lead to epithelial injury and malfunction in giardiasis and other malabsorptive disorders of the intestine.

Intestinal giardiasis. Mini-review / Giardiasis intestinal. Mini-Revisión

Giardia intestinalis is a common parasite in our country and the rest of the world and is responsible for several clinical disturbances that include dysentery type diarrheas, recurrent abdominal pain, duodenitis, jejunitis, cholecystitis and in some cases toxemias and convulsions. In this paper we review recent concepts of intestinal giardiasis, considering the basic aspects of the biology and physiology of Giardia intestinalis, its morphology and its relationship the parasite pathogenicity. We detail the physiopathological mechanisms responsible for the different clinic manifestations of giardiasis, the specific laboratory and endoscopic methods of diagnosis and the most recent advances in the treatment and prophylaxis of this disease.

effect of breast feeding on amoebiasis, giardiasis, and cryptosporidiosis in children below the age of two years

To determine the effect of breast feeding on amoebiasis, giardiasis and cryptosporidiosis in infants in our region. Stool samples were collected from 180 children below 2 years of age who suffered from acute diarrhea. There were 81 breast fed, 49 bottle fed, and 50 mixed bottle and breast fed children. The direct smear method and then formalin-ether sedimentation were carried out to detect intestinal parasites. Fecal smears were prepared from the sediment and stained by the modified Ziehl-Neelsen method for the recovery of acid-fast oocysts of Cryptosporidium. Intestinal amoebiasis, giardiasis and cryptosporidiosis were found at the highest rates [53%, 55% and 64% respectively] among bottle fed children. Collectively, 16.3% of the bottle fed children with diarrhea tested positive for parasitic infections. Breast feeding appears to reduce the risk of these parasitic infections and should be encouraged

Immunofluorescent detection of both giardia lamblia and cryptosporidium parvum using anti-cryptosporidium oocyst antibodies

In the present work, a polyspecific anti-cryptosporidium oocyst antibodies was used for simultaneous detection of both parasites in human stool. Known positive formalinized human stool specimens of Giardia sp. [n = 10], Cryptosporidium sp. [n=7] mixed infection [n=3] and negative specimens [n = 20] were tested using direct fluorescent technique against the developed antibodies. All positive stool samples for Cryptosporidium and 9 out of 10 Giardia samples or each alone showed fluorescence with variable intensities, while no negative sample harbored other parasites had fluorescence. This newly used polyspecific antibodies offer the advantages of screening of a large number of patients, particularly in outbreaks. Additionally, it represents a cheaper alternative for the most sophisticated and costly immunoassay kits using the monoclonal antibodies with more or less the same diagnostic potentials

Giardia duodenalis: Analysis of humoral immune response in experimentally infected gerbils (Meriones unguiculatus)

In this work, we have analyzed the humoral immune response in Mongolian gergils infected with Giardia duodenalis trophozoites of strains P-1 ad WB. The course of infection in the animals was assessed by monitoring cyst shedding in feces, and serum samples were collected at weekly intervals to measure antibody levels by ELISA. Parallel studies were carried out to determine the patterns of total and surface antigens of the parasite recognized by antibodies using Western blot and radioimmunoprecipitation (RIP) assays with the use of homospecific enzyme conjugates. Typical patterns of cyst shedding were observed in the infected animals and cyst numbers per grams of feces were consistently higher in gerbils infected with WB strain. Antibody levels to G. duodenalis antigens were observed by week 2 post-infection and were still detectable 4 months after infection. G. duodenalis antigen showed a complex but quantitatively and qualitatively different recongnition pattern by infection-induced antibodies in Western blot assays which related to infecting strain. However, RIP assays showed a more restricted and common pattern of recognition of surface antigens from either strain. Taken together, the data obtained in this study provides further information regardin direct comparisons among infecting strain, pattern of infectivity, and host immune response toward G duodenalis antigens in the gerbil model

Prevalencia de giardiasis en pacientes con sintomatología digestiva alta Centro Universitario de Salud Pedro P. Díaz julio 95 - junio 96

El presente estudio estuvo orientado a determinar la prevalencia de infección por Giardia lamblia en pacientes del Servicio de Gastroenterología del Centro Universitario de Salud Pedro P. Díaz, que acudieron durante el periodo de un año, de Julio - 95 a Junio - 96, presentando sintomatología digestiva alta. Se estudiaron 130 estudios clínicos-endoscópicos y además el aislamiento de G. lamblia por dos métodos diagnósticos: el examen parasitológico de heces (3 en 1) y examen directo de biopsia duodenal. Encontramos una alta prevalencia de infección por G. lamblia, 40.77 por ciento para ambos métodos. Se confirma la relativa ineficiencia del examen de heces que arrojó una baja frecuencia (10.77 por ciento) en comparación con el método de biopsia duodenal (38.46 por ciento). No encontramos diferencias significativas de prevalencia de infección por sexo, grupos de edad, ni procedencia. La sintomatología que motivo la consulta en los pacientes con Giardia fueron: epigastralgia (75.47 por ciento) meteorismo y flatulencia (47.17 por ciento), dispepsia (26.42 por ciento), baja de peso (26.42 por ciento) y diarrea periodica (24.53 por ciento). En vista de que el espectro de la enfermedad giardiásica es bastante amplio; se señala la importancia del parisitismo como coadyuvante y en algunas oportunidades como causante de la sintomatología de tracto digestivo alto. El hallazgo endoscópico de duodenitis se obtuvo en 32.08 por ciento de pacientes Giardia-positivos y 23.38 por ciento en los Giardia-negativo, no existiendo diferencia estadistica entre ambos grupos pero no se descarta también la posible vinculación etiológica de G. lamblia con esta patología. La alta prevalencia de G. lamblia en pacientes con sintomatología digestiva alta, hallado con el método de biopsia duodenal destaca el valor de la Fibroendoscopía como método diagnóstico eficaz de la giardiasis, por lo que se recomienda su uso en forma rutinaria.

Imunopatologia da giardíase / Pathoimmunology of the giardiasis

Este artigo faz uma revisäo dos mecanismos imunopatogênicos envolvidos na infecçäo por Giardia lamblia, aspectos da interaçäo parasita-hospedeiro e a aplicaçäo deste conhecimento no seu diagnóstico

Effects of protein deficiency and natural intestinal infection with Giardia lamblia on jejunal intraepithelial lymphocytes in rats of different ages

1. The interaction between experimental protein deprivation and natural intestinal infection by Giardia lamblia was studied in terms of its effects on the intraepithelial lymphocyte (IEL) population and morphology of the jejunal mucosa of rats of different ages. 2. Young, adult and old male Wistar rats received a protein-deficient diet (2 casein) or a control diet (20 casein) for 42 days. Mucosal height and the number of lymphocytes located among 500 consecutive epithelial cells (EC) along the villi or crossing the basement membrane were determined in PAS-stained jejunal fragments. 3. The number of IEL increased progressively with animal age, from 14 to 25 per 100 epithelial cells, with significant differences between age ranges. However, the number of IEL did not differ between control and protein-deficient rats in any of the age groups. The proportion of lymphocytes crossing the basement membrane was approximately two-fold greater in young (2.8/100 EC) and adult (5.8/100 EC) protein-deficient animals than in their respective controls (1.6 and 2.8/100 EC). The intensity of parasite colonization was moderate, from 3 to 5/100 EC and did not differ between groups. The pattern of morphologic changes of jejunal mucosa in protozoal infection did not differ between control and protein-deficient animals in any of the three age groups. 4. We conclude that intestinal infection with Giardia lamblia probably stimulated the local immune response, masking the reduction of the IEL population induced by protein deficiency. The increase in lymphocyte numbers with age may be related to prolonged antigenic stimulation promoted by infection.

Prevalencia de parasitismo intestinal en niños supuestamente sanos. Valoración de su inmunidad humoral / Prevalence of intestinal parasitism in child

Se estudió la prevalencia de parasitismo intestinal en un total de 132 niños, clasificados como supuestamente sanos desde el punto de vista gastroentérico, los cuales forman parte de la matrícula de un círculo infantil ubicado en el municipio Habana Vieja. Se recolectaron 3 muestras de heces en cada caso, y se pesquisó, además de otros protozoarios, el Cryptosporidium. También se realizó la cuantificación de inmunoglobulinas G; A; M y E a los casos con Giardia lamblia. La prevalencia obtenida fue de 47,7 por ciento , con predominio de la giardiasis en el 96,8 por ciento de éstos. El 40 por ciento de los niños valorados inmunológicamente presentó algún tipo de déficit, fundamentalmente el de la inmunoglobulina A

Evaluación de la respuesta de anticuerpos en pacientes con giardiasis / Assessment of antibody response in patients with giardiassis

Se estudiaron 104 pacietnes con sintomatología clínica compatible con una giardiasis, de éstos, 101 resultaron parasitológicamente positivos. La presencia de anticuerpos fue demostrada en 99 de ellos, para una sensibilidad de la técnica de inmunofluorescencia indirecta de 96 por ciento y una especificidad de 90 por ciento . La respuesta de anticuerpos fue significativamente mayor (p<0,01) en aquellos pacientes sin antecedentes de la enfermedad, se observó una caída significativa (p<0,01) de los títulos de anticuerpos en todos los pacientes 1 mes posterior al tratamiento eefectivo y la persistencia de títulos positivos en aquéllos que no curan

Serodiagnosis of giardiasis by counterimmunoelectrophoresis and indirect immunofluorescence tests

Serodiagnosis of giardiasis by counterimmunoelectrophoresis assay [CIEP] and indirect immunofluorescence test [IFAT] were evaluated versus stool examination. Giardia Lamblia antibodies were detected in sera of 46.9% and 53.1% using CIEP and IFAT respectively out of 49 giardiasis-infected children [5-10 years] diagnosed microscopically, Among the control group [22 children] 13.6% and 18.2% were positive by CIEP and IFAT respectively. The findings of the study showed that we can not rely on serology to diagnose giardiasis specially in cases with low excretion rates

Characterization of surface associated antigens of axenic Giardia lamblia trophozoites & their recognition by human sera.

Two surface associated antigens (GLSA-82 and GLSA-56) of axenically grown G. lamblia trophozoites (PI strain) were affinity purified from its sonic extract. Both GLSA-82 and GLSA-56 were heat labile, sensitive to treatment with pronase, trypsin and were also sodium metaperiodate modifiable as assessed by micro ELISA. Lectin binding studies revealed that GLSA-82 specifically bound concanavalin A and pokeweed mitogen, and had alpha-methyl mannoside and n-acetyl-B-d-glucosamine sugar moieties. However, GLSA-56 selectively bound Ricinus communis agglutinin and phytohaemagglutinin, and had B-d-galactose and n-acetyl-B-d-galastosamine as sugar moieties. Human sera obtained during acute G. lamblia infection recognised GLSA-82 and GLSA-56 antigens. However, the antibody levels to GLSA-82 were significantly lower (P less than 0.05) during active giardiasis infection. Such surface associated antigens may be target of antiparasitic immune responses and thus, may modulate disease processes.

Relación hospedero-parásito en Giardiasis: (revisión) / Relation parasite-host in Giardiasis: (review)

En Guatemala la giardiasis tiene mucha importancia clínica epidemiológica. En algunas áreas del país su frecuencia alcanza más de un 35/100 en la población general. En este artículo se revisan aspectos relacionados con la fisiopatología de la diarrea producida por este parásito, también se describe nuestra experiencia en la investigación de la respuesta inmune de pacientes infectados y su relación con otros síndromes clínicos como la mala absorción instestinal que se ha atribuído a la infección por Giardia lamblia

Virulence of Giardia lamblia: an in vitro study on host-parasite interaction.

Trophozoites of G. lamblia, a human parasite, were lysed by polymorphonuclear leukocytes (PMNL) of healthy individuals during in vitro interaction. However, the parasite damaged PMNL of giardiasis patients. A prior treatment of giardia trophozoites with anti-giardia serum, caused agglutination of pathogen and, thereby, the cytotoxic capacity of the parasite was reduced. Interaction of giardia-trophozoites with peritoneal macrophage, derived from infected mouse, reduced the phagocytic activity of the latter to 43% (against 100% in control). Macrophage activity was, however, stimulated to 131% when the mice were immunized with giardia antigen prior to experimental infection. Giardia extract proved cytotoxic at a dose of 0.7 mg, to HeLa cells in tissue culture. These in vitro studies offer experimental evidence of the cytotoxic and immuno-toxic behaviour of G. lamblia towards the host cells.