Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal

Experimental infection of Crested Caracara (Caracara plancus) with Toxoplasma gondu simulating natural conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Discontinuation of maintenance therapy against toxoplasma encephalitis in AIDS patients with sustained response to anti-retroviral therapy

Discontinuation of maintenance therapy against toxoplasma encephalitis (TE) for individuals infected with human immunodeficiency virus (HIV) who are receiving successful anti-retroviral therapy is considered safe. Nevertheless, there are few published studies concerning this issue. Within the setting of the Swiss HIV Cohort Study, this report describes a prospective study of discontinuation of maintenance therapy against TE in patients with a sustained increase of CD4 counts to > 200 cells/microL and 14% of total lymphocytes, and no active lesions on cerebral magnetic resonance imaging (MRI). In addition to clinical evaluation, cerebral MRI was performed at baseline, and 1 and 6 months following discontinuation. Twenty-six AIDS patients with a history of TE agreed to participate, but three patients (11%) could not be enrolled because they still showed enhancing cerebral lesions without a clinical correlate. One patient refused MRI after 6 months while clinically asymptomatic. Among the remaining 22 patients who discontinued maintenance therapy, one relapsed after 3 months. During a total follow-up of 58 patient-years, there was no TE relapse among the patients who had remained clinically and radiologically free of relapse during the study. Thus, discontinuation of maintenance therapy against TE was generally safe, but may fail in a minority of patients. Patients who remain clinically and radiologically free of relapse at 6 months after discontinuation are unlikely to experience a relapse of TE.

Heat Shock Protein 90 regulates encystation in Entamoeba

Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely - trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

Chemical contaminants, pathogen exposure and general health status of live and beach-cast Washington sea otters (Enhydra lutris kenyoni)

Analyses of blood and liver samples from live captured sea otters and liver samples from beachcast sea otter carcasses off the remote Washington coast indicate relatively low exposure to contaminants, but suggest that even at the low levels measured, exposure may be indicated by biomarker response. Evidence of pathogen exposure is noteworthy - infectious disease presents a potential risk to Washington sea otters, particularly due to their small population size and limited distribution. During 2001 and 2002, 32 sea otters were captured, of which 28 were implanted with transmitters to track their movements and liver and blood samples were collected to evaluate contaminant and pathogen exposure. In addition, liver samples from fifteen beachcast animals that washed ashore between 1991 and 2002 were analyzed to provide historical information and a basis of reference for values obtained from live otters. The results indicate low levels of metals, butyltins, and organochlorine compounds in the blood samples, with many of the organochlorines not detected except polychlorinated biphenyls (PCBs), and a few aromatic hydrocarbons detected in the liver of the live captured animals. Aliphatic hydrocarbons were measurable in the liver from the live captured animals; however, some of these are likely from biogenic sources. A significant reduction of vitamin A storage in the liver was observed in relation to PCB, dibutyltin and octacosane concentration. A significant and strong positive correlation in vitamin A storage in the liver was observed for cadmium and several of the aliphatic hydrocarbons. Peripheral blood mononuclear cell (PBMC) cytochrome P450 induction was elevated in two of 16 animals and may be potentially related to aliphatic and aromatic hydrocarbon exposure. Mean concentration of total butyltin in the liver of the Washington beach-cast otters was more than 15 times lower than the mean concentration reported by Kannan et al. (1998) for Southern sea otters in California. Organochlorine compounds were evident in the liver of beach-cast animals, despite the lack of large human population centers and development along the Washington coast. Concentrations of PCBs and chlordanes (e.g., transchlordane, cis-chlordane, trans-nonachlor, cis-nonachlor and oxychlordane) in liver of Washington beach-cast sea otters were similar to those measured in Aleutian and California sea otters, excluding those from Monterey Bay, which were higher. Mean concentrations of 1,1,1,- trichloro-2,2-bis(p-chlorophyenyl)ethanes (DDTs) were lower, and mean concentrations of cyclohexanes (HCH, e.g., alpha BHC, beta BHC, delta BHC and gamma BHC) were slightly higher in Washington beach-cast otters versus those from California and the Aleutians. Epidemiologically, blood tests revealed that 80 percent of the otters tested positive for morbillivirus and 60 percent for Toxoplasma, the latter of which has been a significant cause of mortality in Southern sea otters in California. This is the first finding of positive morbillivirus titers in sea otters from the Northeast Pacific. Individual deaths may occur from these diseases, perhaps more so when animals are otherwise immuno-compromised or infected with multiple diseases, but a population-threatening die-off from these diseases singly is unlikely while population immunity remains high. The high frequency of detection of morbillivirus and Toxoplasma in the live otters corresponds well with the cause of death of stranded Washington sea otters reported herein, which has generally been attributable to infectious disease. Washington’s sea otter population continues to grow, with over 1100 animals currently inhabiting Washington waters; however, the rate of growth has slowed over recent years. The population has a limited distribution and has not yet reached its carrying capacity and as such, is still considered at high risk to catastrophic events. (PDF contains 189 pages)

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

Inquérito sorológico para toxoplasmose em equinos na região de Botucatu-SP

The frequency of anti-T.gondii IgG antibodies was evaluated and compared in 253 sera samples of equines by modified agglutination test (MAT) and indirect immunofluorescent antibody test (IFAT). Fifteen samples (5.9%) were positive by IFAT and 32 (12.6%) by MAT. Significant difference between the two methods was found when a cutoff titer of 16 was established. There was no correlation with breed, gender, and age of horses examined.

Prevalência de toxoplasmose em gatos dos Estados de São Paulo e Paraná

Foram avaliados pela reação de imunofluorescência indireta (RIFI) para detecção de anticorpos-IgG anti-Toxoplasma, soros de 191 gatos de 3 diferentes municípios do Estado de São Paulo e um município do Estado do Paraná, enviados ao Serviço de Diagnóstico de Zoonoses, da Faculdade de Medicina Veterinária e Zootecnia, UNESP, Campus de Botucatu. Destes animais, 56 (29,3%) possuíam raça definida e 135 (70,7%) sem raça definida (SRD), sendo 88 (46,1%) fêmeas e 103 (53,9%) machos, com idades variando de dois meses a dezoito anos. Obtiveram-se 37 (19,4%) animais reagentes à RIFI, com títulos de 1/16 (n = 10;27,0%), 1/64 (n = 18;48,6%) e 1/256 (n = 9;24,3%). Não houve associação significativa entre sexo, raça, procedência ou idade (p > 0,05), entretanto, a taxa de infecção foi maior em animais mais velhos, com 13,2% dos animais entre dois meses e três anos de idade, 23,5% entre três e seis anos, 17,6% entre seis e nove anos, 25% entre nove e doze anos, 20% entre doze e quinze anos e 50% entre quinze e dezoito anos de idade.

Construção de um indicador qualitativo (status de saúde) para avaliação da qualidade de saúde animal de propriedades rurais

Aiming at developing a tool for assessing the magnitude of farm animal health, an indicator named herd “animal health status” was built. To illustrate the construction of the indicator, serum samples were taken from 923 goats on 17 goat farms in the state of Sao Paulo, Brazil, where three diseases were evaluated along with their impact percentages: caprine arthritis encephalitis, toxoplasmosis and neosporose – all of an infectious and transmissible nature and the cause of considerable economic losses. The mathematical rationale underlying the building of the “animal health status” indicator ranks properties on a 0 - 100% scale in terms of disease positivity in the herd, with the lowest indicator value indicating the highest cumulative disease frequency (0% = all animals tested positive for the three diseases, 100% = all animals tested negative for all diseases). Anti-T. gondii, anti-N. caninum and anti-CAEV antibodies were tested using indirect immunofluorescence reaction (IIFR ≥ 16), Neospora agglutination test (NAT ≥ 25) and agarose gel immunodiffusion (AGID positive or negative), respectively. The animal health status of the farms ranged from 32.38% to 96.40% according to disease positivity. The animal health status value will have a direct reflection on the production chain both on and off the farm, providing not only great market advantages, due to how the expression of animal health will add value to livestock farms, as well as on the consumer who can check the quality of animal health at the beginning of the production chain.

CASE-CONTROL STUDY OF AN OUTBREAK OF ACUTE TOXOPLASMOSIS IN AN INDUSTRIAL PLANT IN THE STATE OF SÃO PAULO, BRAZIL

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.