A risk infection simulation model for fusarium head blight of wheat

Fusarium Head Blight (FHB) is a disease of great concern in wheat (Triticum aestivum). Due to its relatively narrow susceptible phase and environmental dependence, the pathosystem is suitable for modeling. In the present work, a mechanistic model for estimating an infection index of FHB was developed. The model is process-based driven by rates, rules and coefficients for estimating the dynamics of flowering, airborne inoculum density and infection frequency. The latter is a function of temperature during an infection event (IE), which is defined based on a combination of daily records of precipitation and mean relative humidity. The daily infection index is the product of the daily proportion of susceptible tissue available, infection frequency and spore cloud density. The model was evaluated with an independent dataset of epidemics recorded in experimental plots (five years and three planting dates) at Passo Fundo, Brazil. Four models that use different factors were tested, and results showed all were able to explain variation for disease incidence and severity. A model that uses a correction factor for extending host susceptibility and daily spore cloud density to account for post-flowering infections was the most accurate explaining 93% of the variation in disease severity and 69% of disease incidence according to regression analysis.

Experimentally induced intravaginal Tritrichomonas foetus infection in a mouse model

The interest to develop research on the host-parasite relationship in bovine tritrichomonosis has accomplished the use of experimental models alternative to cattle. The BALB/c mouse became the most appropriate species susceptible to vaginal Tritrichomonas foetus infection requiring previous estrogenization. For the need of an experimental model without persistent estrogenization and with normal estrous cycles, the establishment and persistence of vaginal infection on BALB/c mouse with different concentrations of T. foetus in two experimental groups was evaluated. Group A was treated with 5mg of b-estradiol 3-benzoate to synchronize the estrous, 48 hours before the T. foetus vaginal inoculation, and Group B was inoculated in natural estrus. At 5-7 days after treatment, estrogenic effect decreased allowing all animals to cycle regularly during the experiment. From the first week post-infection, samples of vaginal mucus were taken from all animals during 34 weeks, in order to evaluate the course of infection and the stage of the estrus cycle. Group A showed 93.6% of infected animals, and Group B showed 38%. Different doses of T. foetus were assayed to establish the vaginal infection, with a persistence of 34 weeks. Although different behavior was observed in each subgroup belonging to either Group A or Group B, there were no significant differences among the infecting doses used. The b-estradiol 3-benzoate treatment had a favorable effect on the establishment of the infection (P<0.0001), but it did not influence its persistence (P=0.1097). According to the results, an experimental mouse model is presented, appropriate for further studies on mechanisms of pathogenicity, immune response, protective evaluation of immunogen and therapeutic effect of drugs.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Occurrence of infection with Toxoplasma gondii and factors associated with transmission in broiler chickens and laying hens in different raising systems

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. The aim of the present study was to determine the occurrence and identify the risk factors associated with transmission of T. gondii to chickens raised in different systems (free-ranged and confined) to produce eggs or meat. The 810 animals were allocated in two experimental groups according to the production system purpose: 460 broiler chickens (Group 1) and 350 layer chickens (Group 2). In order to analyze the possible factors involved in T. gondii infection in the chickens, an epidemiological questionnaire was developed for all properties.The serological detection of anti-Toxoplasma gondii antibodies was performed by Indirect Immunofluorescence (IFAT) and by Enzime Linked Imunossorbent Assay (ELISA). Since the agreement index (kappa) between these two serological techniques was considered high, 21.2% of the 810 animals were considered reactive. In Group 1, 12.2% (56/460) were positive, while in the Group 2 the positivity rate was 33.1% (116/350). The production system may be influencing the seropositivity of the animals in both groups. However, only in Group 2 it was possible to notice a statistically significant relationship between the breeding system and the frequency of positive sera. This result indicates that, at least for laying hens, the production system is directly involved in T. gondii infection. The contact with cats in Group 1 did not influence the distribution of seroreactive animals, but in Group 2 a significant relationship was observed. The occurrence of anti-T. gondii antibodies was high in both groups (broiler and posture chickens). Free-ranged chickens raised for egg production proved to be the most exposed group to the T. gondii infection. This can be related to the fact that these animals stay for longer periods in the farms, in direct contact with possibly contaminated soil by the presence of domestic cats.

Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.

Screening of resistance to egyptian broomrape infection in tomato varieties

Parasitic weed species of the genus Orobanche are serious threat for the production of several crops in Europe, Africa and Asia. Research on resistant host plant varieties is one of the most effective management strategies for this parasitic weed. In this study, the susceptibility of twenty-nine tomato varieties to broomrape infection (Orobanche aegyptiaca) under greenhouse conditions was investigated. The employed experimental design was completely randomized with three replications. Differences in susceptibility to infection were monitored among tomato varieties based on their difference in the number of emerged shoots of broomrape and broomrape dry weight (shoots and tubercles). Date of Orobanche emergence varied over a period of 3 to 30 days between varieties. Very late infection was monitored for varieties of Cal-jN3, Viva, Caligen 86, Packmor, CSX 5013, Hyb. PS 6515 and Hyb Petopride5. Differences in the growth and fruit yield among tomato varieties were also found in response to broomrape infestation. Moderate levels of resistance were obtained in Viva, Caligen 86, Hyb. PS 6515, Hyb.Firenze (PS 8094) and Cal-jN3 among other tomato varieties. In contrast, varieties of Kimia-Falat, Hyb. Petopride II and Hyb.AP865 were the most susceptible hosts to Orobanche aegyptiaca.

An experimental model for the measurement of renal function after temporary ureteral obstruction in the rat

We present the results obtained with a ureterovesical implant after ipsilateral ureteral obstruction in the rat, suitable for the study of renal function after deobstruction in these animals. Thirty-seven male Wistar rats weighing 260 to 300 g were submitted to distal right ureteral ligation and divided into 3 groups, A (N = 13, 1 week of obstruction), B (N = 14, 2 weeks of obstruction) and C (N = 10, 3 weeks of obstruction). The animals were then submitted to ureterovesical implantation on the right side and nephrectomy on the left side. During the 4-week follow-up period serum levels of urea and creatinine were measured on the 2nd, 7th, 14th, 21st and 28th day and compared with preoperative levels. The ureterovesical implantation included a psoas hitch procedure and the ureter was pulled into the bladder using a transvesical suture. During the first week of the postoperative period 8 animals died, 4/13 in group A (1 week of obstruction) and 4/14 in group B (2 weeks of obstruction). When compared to preoperative serum levels, urea and creatinine showed a significant increase (P<0.05) on the 2nd postoperative day in groups A and B, with a gradual return to lower levels. However, the values in group B animals were higher than those in group A at the end of the follow-up. In group C, 2/10 animals (after 3 weeks of obstruction) were sacrificed at the time of ureterovesical implantation due to infection of the obstructed kidneys. The remaining animals in this group were operated upon but all of them died during the first week of follow-up due to renal failure. This technique of ureterovesical implantation in the rat provides effective drainage of the upper urinary tract, permitting the development of an experimental model for the study of long-term renal function after a period of ureteral obstruction

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

Cytokine profiles during experimental Chagas' disease

People infected with Trypanosoma cruzi remain so for life, yet only 30-40% of these individuals develop characteristic chagasic cardiomyopathies. Similarly, when infected with the Brazilian strain of T. cruzi, DBA/2 mice develop severe cardiac damage while B10.D2 mice do not. To better understand the immunological parameters that may be involved in the disease process, we have used this murine model (DBA/2 vs B10.D2) and compared the changes in cytokine production during the course of infection with T. cruzi. Concanavalin A (Con A) stimulation of spleen cells harvested during the acute phase (day 30) resulted in similarly high levels of IFN-g in both mouse strains. However, the amount of IFN-g in supernatants from cultures of B10.D2 spleen cells initiated during the chronic phase (day 72) was at subacute levels, whereas secretion by chronic DBA/2 spleen cells remained high. In addition, Con A-stimulated spleen cells from acute DBA/2 mice produced approximately twice as much IL-10 and significantly more IL-4 than cells from B10.D2 mice. IL-4 secretion remained low by cells from chronic B10.D2 mice, but when using cells from chronic DBA/2 mice, levels continued to increase beyond the already high levels secreted by cells harvested during the acute phase. Proliferative responses to Con A stimulation by spleen cells from DBA/2 mice were significantly higher than those from B10.D2 mice in both the acute and chronic phases. These data suggest that enhanced responses in DBA/2 mice, which may be related to a higher parasite burden, a lack of down-regulation, and/or the onset of autoimmune phenomena, correlate with the more severe cardiomyopathy seen in pathopermissive mice.

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

Urethroplasty using a bovine pericardium graft: an experimental study using normal urethras from dogs

The use of bovine pericardium as a urethral patch to substitute a ventral segment of canine urethras was studied. Healing, epithelial growth, urethral permeability, fistulas, and calcification were analyzed. Thirty male mongrel dogs of medium and large size underwent resection of a ventral segment of the medial urethra measuring 2.0 x 0.5 cm, which was replaced with a bovine pericardium graft, treated with buffered glutaraldehyde and preserved in formaldehyde. Two running sutures of polygalactin 5-0 were applied, one on each side of the patch. The corpus spongiosum was closed with uninterrupted suture and the skin with interrupted suture of polygalactin 5-0. Six months later, the animals were examined and sacrificed under anesthesia. Retrograde urethrograms showed that the urethral healing was complete in six of the 30 animals, without stenosis, fistulas or dilations. Microscopic examination showed complete epithelization of these six urethras. The remaining 24 animals presented urethrocutaneous fistulas without stenosis, demonstrated by urethral catheterism using a 10-Fr plastic catheter. These data show that a successful urethral reconstruction of the penile urethra was possible in only 20% of the operated animals. Infection and leakage may be the cause of the urethrocutaneous fistulas present in 80% of cases. Further studies are necessary to determine whether such fistulas are avoidable. If they are, the bovine pericardium may well be an option in the treatment of urethral lesions in dogs.

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

Immunity and immunosuppression in experimental visceral leishmaniasis

Leishmaniasis is a disease caused by protozoa of the genus Leishmania, and visceral leishmaniasis is a form in which the inner organs are affected. Since knowledge about immunity in experimental visceral leishmaniasis is poor, we present here a review on immunity and immunosuppression in experimental visceral leishmaniasis in mouse and hamster models. We show the complexity of the mechanisms involved and differences when compared with the cutaneous form of leishmaniasis. Resistance in visceral leishmaniasis involves both CD4+ and CD8+ T cells, and interleukin (IL)-2, interferon (IFN)- gamma, and IL-12, the latter in a mechanism independent of IFN- gamma and linked to transforming growth factor (TGF)-ß production. Susceptibility involves IL-10 but not IL-4, and B cells. In immune animals, upon re-infection, the elements involved in resistance are different, i.e., CD8+ T cells and IL-2. Since one of the immunopathological consequences of active visceral leishmaniasis in humans is suppression of T-cell responses, many studies have been conducted using experimental models. Immunosuppression is mainly Leishmania antigen specific, and T cells, Th2 cells and adherent antigen-presenting cells have been shown to be involved. Interactions of the co-stimulatory molecule family B7-CTLA-4 leading to increased level of TGF-ß as well as apoptosis of CD4+ T cells and inhibition of macrophage apoptosis by Leishmania infection are other components participating in immunosuppression. A better understanding of this complex immune response and the mechanisms of immunosuppression in experimental visceral leishmaniasis will contribute to the study of human disease and to vaccine development.

IgE expression on the surface of B1 and B2 lymphocytes in experimental murine schistosomiasis

In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.