Microsatellite-primed PCR and random primer amplification polymorphic DNA for the identification and epidemiology of dermatophytes.

This study demonstrates the capacity of the one-step polymerase chain reaction (PCR) fingerprinting method using the microsatellite primers (GACA)4 or (GTG)5 (MSP-PCR) to identify six of the most frequent dermatophyte species causing cutaneous mycosis. PCR with (GACA)4 was a suitable method to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale among 82 Argentinian clinical isolates, producing the most simple and reproducible band profiles. In contrast, the identification of Trichophyton mentagrophytes and Trichophyton tonsurans was achieved using PCR with (GTG)5. In this way, the sequential application of PCR using (GACA)4 and (GTG)5 allowed the successful typification of clinical isolates which had not been determined by mycological standard techniques. In this work, the intraspecies variability among 33 clinical isolates of M. canis was detected using random amplification of polymorphic DNA (RAPD-PCR) with the primers OPI-07 and OPK-20. The genetic variations in the isolates of M. canis were not associated with clinical features of lesions or pet ownership, but a geographical restriction of one genotype was determined with OPK-20, suggesting a clonal diversity related to different ecological niches in certain geographical areas. The results of this work demonstrate that the detection of intraspecies polymorphisms in M. canis by RAPD-PCR may be applied in future molecular epidemiological studies to identify endemic strains, the route of infection in an outbreak or the coexistence of different strains in a single infection.

Immunization with crude antigens plus aluminium hydroxide protects cattle from Fasciola hepatica infection.

The ability of total homogenate (TH) of Fasciola hepatica conjugated with aluminium hydroxide (alum) or Freund's complete adjuvant (FCA) to protect cattle against experimental fasciolosis was evaluated. Compared with the infected group, the immunized animals with alum-TH and FCA-TH presented a significant reduction in fluke burden (85.9% and 96.8%, respectively), a higher percentage of short-sized worms, a marked reduction in the released eggs in faeces (89% and 57%, respectively), as well as an increased production of specific antibodies before infection. The alum-TH immunized group also showed a significant increase in the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) as early as 4 weeks before infection. Although both immunized groups (alum-TH and FCA-TH) were able to develop an efficient protective immune response to metacercarial challenge, an earlier PBMC response, lower hepatic damage and less effect on weight gain were found in alum-immunized animals. Therefore, alum is a good candidate for future immunization against bovine fasciolosis.

Inhibition of spleen cell proliferative response to mitogens by excretory-secretory antigens of Fasciola hepatica.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) on the proliferative response of spleen mononuclear (SpM) cells of normal rats to stimulation with mitogens has been examined. When ESA was added to normal SpM cells, there was a decrease in the proliferative response to concanavalin A (Con A) or lipo-polysaccharide (LPS) in a dose-dependent manner. The addition of indomethacin, which blocks prostaglandin synthesis, or N omega-nitro-L-arginine methyl ester (L-NAME) a specific inhibitor of nitric oxide (NO) synthase, had no effect on the ability of ESA to suppress the proliferative response to Con A. However, supplementation of the culture media with catalase, which degrades hydrogen peroxide (H2O2) or superoxide dismutase (SOD) to remove superoxide anion (O2), resulted in a restoration of proliferation to Con A. When LPS was used as mitogenic stimulus no inhibitor added to the culture restored the proliferation. These results suggest that H2O2 and O2- are involved in the suppressor phenomenon induced by ESA in the T-cell proliferative events.

Cytokines involved in the immunosuppressor period in experimental fasciolosis in rats.

The aim of this study was to evaluate the kinetics of the cytokines interferon-gamma, interleukin-2, interleukin-10 and interleukin-4 produced by spleen mononuclear cells stimulated by Con A during an experimental infection in rats with Fasciola hepatica. The proliferative response to Con A of Spm cells from rats infected with F. hepatica was significantly decreased on day 7 post-infection (P<0.006) and simultaneously an increase of interferon-gamma, interleukin-10 and interleukin-4 production along with a decrease of interleukin-2 by spleen mononuclear cells were observed. Interleukin-4 and interleukin-10 were involved in ablating cellular proliferation in vitro, as the addition of neutralising antibodies to either cytokine reversed the proliferative block. The addition of exogenous recombinant interleukin-2 also restored the proliferative response by spleen mononuclear cells obtained 7 days after infection from infected rats. At the same time, we found an increase in interleukin-10 production by peritoneal cells (in close contact with the flukes) and decreased nitric oxide levels. In addition, histological studies on the liver on day 7 after infection showed the presence of parasite inside migratory tunnels in the parenchyma, and polymorphonuclear leukocytes, predominantly eosinophils, around the parasite. The transient suppression in proliferative response mediated by cytokines interleukin-4 and interleukin-10 in the spleen, and diminution of nitric oxide production in the peritoneum could be mechanisms to evade the protective immune response during the first stages of liver penetration by the parasite.

Immunobiological effects of AFB1 and AFB1-FB1 mixture in experimental subchronic mycotoxicoses in rats.

Maize co-contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is frequently found in several countries. Although the alterations on nutritional and immunologic parameters induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced in animals by a subchronic administration of both toxins mixtures. We have studied the nutritional and immunological alterations induced in rats fed during 90 days with a diet without mycotoxins, containing 40 ppb AFB1, and with a diet containing a mixture of 40 ppb AFB1 and 100 ppm FB1. Animals fed with the mixture of toxins obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMC) in vivo was higher in animals fed with AFB1. In in vitro studies, lower proliferations of SMC pre-exposed to AFB1 and to the mixture of toxins were detected. The SMC of animals fed with AFB1 produced lower levels of IL-2, higher of IL-4 and equal levels of IL-10. The SMC of animals fed with both toxins produced higher levels of IL-4, lower of IL-10 and equal levels of IL-2. The SMC preincubated with an AFB1-FB1 mixture produced higher concentrations of IL-4, lower of IL-10 and equal levels of IL-2. The peritoneal macrophages of animals that consumed AFB1 released less H(2)O(2), while animals fed with the mixture of toxins produced higher levels. In in vitro studies, macrophages pre-exposed to the mixture of toxins released less H(2)O(2). These results show different immunobiological effects produced by a mixture of mycotoxins in comparison to the individual action of the same toxins.

Modification of accessory activity of peritoneal cells from Fasciola hepatica infected rats.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) was studied in the modulation of the accessory functions of peritoneal cells (PC) of rats infected with the parasite. PC rats infected with F. hepatica 7 and 14 days previously showed a marked decrease in phagocytic activity against Candida tropicalis (P < 0.007 and P < 0.004, respectively). The same effect was observed when the assay was carried out with PC from animals injected 7 days before with ESA (P < 0.001) including PC previously treated in vitro with ESA. To investigate the effect of ESA on the antigen presenting ability, PC of animals infected before transfer to syngeneic normal rats were stimulated in vitro with either F. hepatica whole antigen (FhWA), ESA or human serum albumin (HSA). Delayed type hypersensitivity (DTH) response to the different antigens studied over a 35 day period was negative in rats transferred with sensitised PC. When these animals were immunised with the corresponding antigen the DTH response became positive. A similar result was obtained in PC receptors from ESA inoculated rats and in vitro stimulated with FhWA or HSA. These data suggest that the alterations observed in the functions of peritoneal cells may, in part be due to the effect of the F. hepatica ESA.

Experimental subchronic mycotoxicoses in mice: individual and combined effects of dietary exposure to fumonisins and aflatoxin B1.

We have used a murine model of subchronic mycotoxicoses produced by ingestion of mycotoxins. The five groups of animals studied were fed for 30, 60 and 90 days, respectively, with commercial diet (CD), experimental control diet (ECD), experimental with fumonisin B1 diet (EFD) and experimental with mixtures of mycotoxins diet (EMD). The animals fed EFD and EMD showed a significant increase in feed consumption/day with respect to the animals fed ECD (P < 0.005 for both groups). The biochemical measurements showed significant differences at 90 days in those animals fed EAD exhibiting a marked decrease in the values of alkaline phosphatase (ALP) and cholesterol (P < 0.05), along with a significant increase in calcium (P < 0.01). Differences in the decrease of the parameters studied were observed in mice fed EFD for triglycerides, cholesterol and calcium (P < 0.05 for all of them). The activity of aspartate transaminase (AST) increased significantly in animals fed EMD (P < 0.01). The tissue specimens at 60 days showed lesions in the livers of the animals fed EAD and EFD. At 90 days, and in those fed EAD, EFD and EMD, the lesions were intensified in the liver at 60 days in 80, 90 and 100% of the animals, respectively.

Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses.

Normal rats i.p. injected with Fasciola hepatica excretor-secretor antigen (ESA) induced a population of spleen mononuclear (SpM) cells, which suppressed the delayed type hypersensitivity (DTH) response to parasite antigens as well as to non-related antigens (human serum albumin) by adoptive transfer. A similar effect was observed when the cell transfer was performed with SpM cells non-adherent to nylon wool. The DTH was not modified by cells transfer adherent to nylon wool in syngeneic receptor animals. The observed suppression depended on the concentration and inoculation moment of the antigen; 1.8 mg of protein ESA being enough to suppress the DTH response at the different days studied, before and after immunization with whole F. hepatica antigens. A marked suppression was observed when ESA was injected on day 7 pre-immunization. On the other hand, inoculation of ESA treated with 0.01 M sodium periodate (carbohydrate oxidant) diminished the suppressor effect found after the native ESA inoculation, indicating participation of ESA glucidic components in induced suppression. Inoculation of ESA fractions obtained from polyacrylamide gel elution with different MW range, showed that components between 12 and 23 kDa actively induced suppression to the DTH response to parasite antigens.

Serological, electrophoretic and biological properties of Fasciola hepatica antigens.

Fasciola hepatica somatic antigen, its partially purified fractions and excretion-secretion products were investigated as to serological, electrophoretic and biological properties. In a Sephadex G-100 column (SG-100), Fasciola hepatica total antigen (FhTA) gave 5 fractions, and SDS-PAGE analysis showed they were glycoproteins ranging from 14 to 94 kDa molecular weight (MW). When these fractions were analyzed by enzyme-linked immunotransfer blot (EITB) and immunodiffusion in gel (ID) with serum from immunized rats with FhTA, the presence of different antigenic components was revealed. In the SDS-PAGE of excretor-secretor antigen (ESA), it was possible to observe peptides from 12 to 22 kDa, which were also present in FhTA. When the FhTA, its fractions and the ESA were analyzed by EITB with the immune rat serum (IRS), it was observed that only some fractions of the SG-100 shared antigens with the FhTA and ESA. Moreover, DTH and ITH responses were studied in FhTA immunized rats challenged with these different antigen components, revealing that the protein/carbohydrate ratio is important for inducing DTH response. The ESA was the most active component in the DTH and ITH response.

Possibility of in-hospital infection by Cryptococcus neoformans in patients with AIDS.

The objective of the present work was to carry out a survey of soil samples taken from different areas of a hospital of infectious disease located in the city of Córdoba, where three AIDS patients were hospitalized during different periods in the same ward. The three of them returned with meningeal cryptococcosis between three or five months after having been discharged. Cryptococcus neoformans was isolated in 8/10 samples collected outside the hospital, near the pigeon house. The samples collected from the AIDS patients ward and its surroundings were negative. These findings suggest that the patients may have been infected by the fungus during their first stay in hospital.

Fasciola hepatica-induced immune suppression of spleen mononuclear cell proliferation: role of nitric oxide.

The aim of the present study was to evaluate the proliferative response of spleen mononuclear cells (Spm) to mitogens in rats infected with Fasciola hepatica and its correlation with Spm and peritoneal cell (PC) nitric oxide (NO) production on Days 1, 3, 7, 14, 30, and 60 postinfection. In addition, histological changes in the liver were also studied. The proliferative response to Con A of F. hepatica-infected Spm was significantly decreased on Day 7 postinfection (P < 0.01). However, a pronounced increase of the proliferative response was detected from Day 3 until Day 60 when Spm were stimulated with LPS. In order to determine whether NO levels were modified during F. hepatica infections, we quantified nitrite in Spm and PC supernatants in cultures. Our results indicate a profound decrease of nitrite production by LPS-stimulated PC on the first and second weeks postinfection, and an increase in the levels of this mediator on LPS-stimulated Spm at the same postinfection time. The F. hepatica excretory-secretory antigen (ESA) was in part involved in the decrease of nitrite production by LPS-stimulated PC. A mechanism to avoid an immune response during the first stages of liver penetration could explain the transient suppression observed in Spm proliferative responses. On the other hand, the decrease in NO production by rat-infected PC could also be one of the strategies of the parasite to avoid the potential killing effect of NO during peritoneal migration.

Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.

The excretory-secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 micrograms/ml of ESA protein (P < 0.03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P < 0.04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.

Experimental cryptococcosis in guinea pigs.

A guinea pig model was used to evaluate immune response to Cryptococcus neoformans. This model shared characteristics with cryptococcosis in humans. Twenty five guinea pigs injected intraperitoneally with 10(7) viable C. neoformans cells developed disseminated disease. Forty days after infection all guinea pigs were killed and autopsy performed. C. neoformans growth in the lungs, brains, livers and spleen of the infected animals were determined. Furthermore, the immune response was characterized by moderate degree of delayed-type hypersensitivity and humoral response. In some organs was observed neutrophil and lymphocyte infiltration with presence of cryptococci cells. The infiltration observed in the organs was probably a consequence of an immune reaction.

Immunosuppression in experimental cryptococcosis in rats. Induction of thymic suppressor cells.

The presence of the microorganism, cortical hyperplasia and germinal centers was detected in the thymus of rats infected with 10(7) viable Cryptococcus neoformans cells and immunized at 7 days afterwards with 2.5 mg (0.1 ml) of human serum albumin (HSA) incorporated to complete Freund's adjuvant (CFA) (Group 2). There was no modification of the glandular structure in the thymus of the animals only immunized with HSA-CFA (Group 1). The weight of the thymus of group 2, animals infected and immunized, was increased compared with the weight of the thymus of group 1 animals, this became evident by the increase of the thymic index (TI) (p less than 0.005). This rate was obtained calculating the thymus weight/total body weight ratio x 1000. Thymic cells (10(7) cells in 1 ml) obtained from both groups of animals were transferred to normal syngeneic rats of the same sex. The recipient rats were immunized with HSA-CFA 24 h later and 14 days after the transference, the response of delayed-type hypersensitivity (DTH) was studied in them. It could be observed that the thymic cells coming from group 2 animals were capable of suppressing significatively the afferent pathway of the DTH response to HSA when compared with the response of the animals that received cells coming from group 1 rats (p less than 0.0001).

Immunosuppression in experimental cryptococcosis in rats. Induction of efferent T suppressor cells to a non-related antigen.

Using a rat model, we have previously demonstrated that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a non-related antigen, human serum albumin (HSA), that has been injected 7 days after the infection. We previously determined that the cryptococcal infection induces afferent suppressor or suppressor induction cells (Ts1) to HSA. The primary objective of the present study was to investigate the suppressor cells involved in the efferent phase of delayed-type hypersensitivity (DTH) response to HSA in rats infected with C. neoformans and immunized with the non-related antigen and determine the role that the Ts1 cell plays in the induction of that cell. For this purpose, the spleen mononuclear (SpM) cells containing the Ts1 or SpM cells from immunized non-infected rats (used as donor controls) were transferred to two groups of syngeneic naive recipients (first recipients). Later, the SpM cells from both groups of animals were transferred to rats immunized with HSA (second recipients). The efferent limb of the DTH response to HSA was suppressed in the recipients that received SpM cells from donors injected with Ts1 cells. Additional HSA antigen was not required for induction of these efferent suppressor cells. Furthermore, we here show that these cells are resistant to treatment with cyclophosphamide (Cy), and that they can activate another suppressor population. The latter are Cy sensitive and are present in the immune recipient.

Experimental coccidioidomycosis: effects of cyclophosphamide in immunologic responses.

Rats were infected with Coccidioides immitis and injected with cyclophosphamide three days pre or post infection. Administration of the drug before the infection caused enhancement of DTH response and decrease of the colony forming units (CFU). Conversely, injection of the drug three days post infection produced contrary effects, indicating that a normal T-cell function is essential as a defense mechanism in C. immitis infection.

Immunosuppression in experimental cryptococcosis in rats. Induction of afferent T suppressor cells to a non-related antigen.

To demonstrate the nature of the suppressor cells elicited in rats infected with Cryptococcus neoformans and immunized with human serum albumin (HSA), spleen mononuclear (SpM) cells were fractionated through a nylon wool column. The adherent and non-adherent populations were collected and transferred to syngeneic rats. In all cases, the non-adherent or T-enriched cells adoptively transferred suppression to HSA, however, the suppressive effects of the non-adherent cells were never as great as those of the unpassed population of SpM cells. The fractions adherent to nylon wool also diminished the delayed-type hypersensitivity response to HSA although this was not significant, but glass-adherent cells did exhibit significant suppressor activity. Immunized, non-infected rats were used as donor controls. Furthermore, we showed that the T-enriched-cells are sensitive to treatment with low doses of cyclophosphamide and that they bind HSA. These data indicate that immune suppression of the induction of the delayed-type hypersensitivity response to HSA in cryptococcosis can occur as a result of infection with C. neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells. Characterization of the non-adherent cells indicates that they are Ts1 cells.

Effect of cyclophosphamide on rats experimentally infected with Cryptococcus neoformans.

This study was undertaken to establish the function of T-lymphocytes in protective immunity against a cryptococcal infection in animals treated with Cyclophosphamide (Cy) pre or post infection and to determine how they relate to the progression of the disease. Inbred Suquia rats were infected either intranasally (i.n.) or intraperitoneally (i.p.) with 10(5) viable Cryptococcus neoformans cells. The infected rats were divided in three groups. One of the groups (group I) was utilized as a control. The second group (group II) was treated with Cy 3 days before the infection. The third group (group III) was treated with Cy 3 days after the infection. At approximately 22 days post infection, C. neoformans growth in selected organs of all animals were determined. In addition, humoral and delayed-type hypersensitivity (DTH) response were assayed in the rats. When the Cy was applied after the infection the DTH was significantly diminished and inverse to the colony forming unit (CFU) which increased leading to the animals death. On the other hand, injection of the drug 3 days before infection did not modify the response, that was comparable in both treated and the control animals. In this study it were found haemagglutinating antibodies in sera from i.n. and i.p. infected rats although at minimal levels and were not present in all animals. The results show that with a low T-cell function induced as a consequence of injecting Cy after the infection, rats did not develop a normal DTH response to cryptococcal infections and were not able to control a cryptococcal infection as well as animals with normal T-cell function.

Immunosuppression in experimental cryptococcosis: variation of splenic and thymic populations and expression of class II major histocompatibility complex gene products.

Previous studies from our laboratory have shown that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a nonrelated antigen, the human serum albumin (HSA). In the present study, we determined the variation of thymus and spleen cell populations in rats infected with C. neoformans and immunized with HSA-CFA at the time when suppressor activity was demonstrated. At 21 days postinfection, the number and the percentage of CD4+CD8+ cells were significantly increased in the thymus together with a minor imbalance in other thymocytes subsets. The study of two class II molecules encoded within the major histocompatibility complex, IA and IE, showed that the total number of class II IA-positive cells was increased in the glands of animals infected when compared to the glands of animals only immunized, while the corresponding percentages were lower than those in control rats. On the contrary a significant increase in both the number and the percentage of IE phenotype was observed in the thymus of infected rats, compared to the animals that were only immunized and used as a control. The IE/IA phenotype ratio within each group was increased in rats injected with the fungus. The study of spleen populations revealed an increase in CD4+ and CD8+ cells and a decrease in the B cells. The IE antigen was increased in the spleen of infected animals. The IA molecule expression showed no difference between the infected animals and the control groups. The IE/IA phenotypes ratio was mildly increased in the spleen of infected rats. These findings reveal that the cryptococcal infection can render an important imbalance in the thymus and spleen T-cell compartment together with a significant increase in the expression of the IE molecule at the time when the suppressor activity was demonstrated in this model.