Protective Effect of Chronic Schistosomiasis in Baboons Coinfected with Schistosoma mansoni and Plasmodium knowlesi.

Malaria and schistosomiasis coinfections are common, and chronic schistosomiasis has been implicated in affecting the severity of acute malaria. However, whether it enhances or attenuates malaria has been controversial due the lack of appropriately controlled human studies and relevant animal models. To examine this interaction, we conducted a randomized controlled study using the baboon (Papio anubis) to analyze the effect of chronic schistosomiasis on severe malaria. Two groups of baboons (n = 8 each) and a schistosomiasis control group (n = 3) were infected with 500 Schistosoma mansoni cercariae. At 14 and 15 weeks postinfection, one group was given praziquantel to treat schistosomiasis infection. Four weeks later, the two groups plus a new malaria control group (n = 8) were intravenously inoculated with 10(5) Plasmodium knowlesi parasites and monitored daily for development of severe malaria. A total of 81% of baboons exposed to chronic S. mansoni infection with or without praziquantel treatment survived malaria, compared to only 25% of animals infected with P. knowlesi only (P = 0.01). Schistosome-infected animals also had significantly lower parasite burdens (P = 0.004) than the baboons in the P. knowlesi-only group and were protected from severe anemia. Coinfection was associated with increased spontaneous production of interleukin-6 (IL-6), suggesting an enhanced innate immune response, whereas animals infected with P. knowlesi alone failed to develop mitogen-driven tumor necrosis factor alpha and IL-10, indicating the inability to generate adequate protective and balancing immunoregulatory responses. These results indicate that chronic S. mansoni attenuates the severity of P. knowlesi coinfection in baboons by mechanisms that may enhance innate immunity to malaria.

Protective Potential of Antioxidant Enzymes as Vaccines for Schistosomiasis in a Non-Human Primate Model.

Schistosomiasis remains a major cause of morbidity in the world. The challenge today is not so much in the clinical management of individual patients, but rather in population-based control of transmission in endemic areas. Despite recent large-scale efforts, such as integrated control programs aimed at limiting schistosomiasis by improving education and sanitation, molluscicide treatment programs and chemotherapy with praziquantel, there has only been limited success. There is an urgent need for complementary approaches, such as vaccines. We demonstrated previously that anti-oxidant enzymes, such as Cu-Zn superoxide dismutase (SOD) and glutathione S peroxidase (GPX), when administered as DNA-based vaccines induced significant levels of protection in inbred mice, greater than the target 40% reduction in worm burden compared to controls set as a minimum by the WHO. These results led us to investigate if immunization of non-human primates with antioxidants would stimulate an immune response that could confer protection as a prelude study for human trials. Issues of vaccine toxicity and safety that were difficult to address in mice were also investigated. All baboons in the study were examined clinically throughout the study and no adverse reactions occurred to the immunization. When our outbred baboons were vaccinated with two different formulations of SOD (SmCT-SOD and SmEC-SOD) or one of GPX (SmGPX), they showed a reduction in worm number to varying degrees, when compared with the control group. More pronounced, vaccinated animals showed decreased bloody diarrhea, days of diarrhea, and egg excretion (transmission), as well as reduction of eggs in the liver tissue and in the large intestine (pathology) compared to controls. Specific IgG antibodies were present in sera after immunizations and 10 weeks after challenge infection compared to controls. Peripheral blood mononuclear cells, mesenteric, and inguinal node cells from vaccinated animals proliferated and produced high levels of cytokines and chemokines in response to crude and recombinant antigens compared with controls. All together, these data demonstrate the potential of antioxidants as a vaccine in a non-human primate model.

Parasite accumulation in placenta of non-immune baboons during Plasmodium knowlesi infection.

BACKGROUND: Placental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease. METHODS: Ten pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by Plasmodium knowlesi blood-stage parasites through the femoral vein on 160(th) day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn's Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman's rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant. RESULTS: Levels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score (rs = 0.7650, P <0.05) and inflammatory score (rs = 0.8590, P <0.05). Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons. CONCLUSION: This study has demonstrated accumulation of parasitized red blood cells and infiltration of inflammatory cells in the placental intravillous space (IVS) of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy.

Influence of trypanocidal therapy on the haematology of vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense.

The aim of this study was to characterise the sequential haematological changes in vervet monkeys infected with Trypanosoma brucei rhodesiense and subsequently treated with sub-curative diminazene aceturate (DA) and curative melarsoprol (MelB) trypanocidal drugs. Fourteen vervet monkeys, on a serial timed-kill pathogenesis study, were infected intravenously with 10(4) trypanosomes of a stabilate T. b. rhodesiense KETRI 2537. They were treated with DA at 28 days post infection (dpi) and with MelB following relapse of infection at 140 dpi. Blood samples were obtained from the monkeys weekly, and haematology conducted using a haematological analyser. All the monkeys developed a disease associated with macrocytic hypochromic anaemia characterised by a reduction in erythrocytes (RBC), haemoglobin (HB), haematocrit (HCT), mean cell volume (MCV), platelet count (PLT), and an increase in the red cell distribution width (RDW) and mean platelet volume (MPV). The clinical disease was characteristic of human African trypanosomiasis (HAT) with a pre-patent period of 3 days. Treatment with DA cleared trypanosomes from both the blood and cerebrospinal fluid (CSF). The parasites relapsed first in the CSF and later in the blood. This treatment normalised the RBC, HCT, HB, PLT, MCV, and MPV achieving the pre-infection values within two weeks while RDW took up to 6 weeks to attain pre-infection levels after treatment. Most of the parameters were later characterised by fluctuations, and declined at one to two weeks before relapse of trypanosomes in the haemolymphatic circulation. Following MelB treatment at 140 dpi, most values recovered within two weeks and stabilised at pre-infection levels, during the 223 days post treatment monitoring period. It is concluded that DA and MelB treatments cause similar normalising changes in the haematological profiles of monkeys infected with T. b. rhodesiense, indicating the efficacy of the drugs. The infection related changes in haematology parameters, further characterise the vervet monkey as an optimal induced animal model of HAT. Serial monitoring of these parameters can be used as an adjunct in the diagnosis and prognosis of the disease outcome in the vervet monkey model.

Schistosome-induced pathology is exacerbated and Th2 polarization is enhanced during pregnancy.

UNLABELLED: THE AIM of this study was to investigate the immunopathological impact of pregnancy on an ongoing experimental schistosomiasis infection. MATERIALS AND METHODS: Female BALB/c mice were randomly divided into three groups (A, B and C) of 15 animals each. The mice in Groups A and B were infected with 40 S. mansoni cercariae, percutaneously. Six weeks post-infection, the mice in Groups B and C (schistosome-naive controls) were mated. Schistosome-induced morbidity and cytokine recall responses were subsequently evaluated at weeks 7 and 8 post-infection. RESULTS: Hepatic and pulmonary lesions resulting from trapped schistosome eggs were more frequent and more severe in Group B mice than in Group A mice. Group C mice had suppressed mitogen-stimulated interleukin 4 (IL-4) but maintained high intereferon gamma (IFN-gamma) responses. In contrast, Group A mice had elevated mitogen- and parasite-specific IL-4 but muted IFN-gamma responses. Group B mice had an early (week 7) high IL-4 response, even higher than in group A mice. CONCLUSION: Taken together the data suggest that pregnancy exacerbates schistosome-induced morbidity, probably through up-regulation of parasite-specific IL-4.

IL-10 is up regulated in early and transitional stages in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense.

IL-10 has been suggested as a possible parameter for human African trypanosomiasis stage determination. However, conclusive experimental studies have not been carried out to evaluate this, which is a prerequisite before a potential test can be validated in humans for diagnostic purposes. We used the vervet monkey model of trypanosomiasis to scrutinize IL-10 in blood and cerebrospinal fluid (CSF). Five adult males were experimentally infected with T. b. rhodesiense. The infected animals became anemic and exhibited weight loss. Parasitemia was patent after 3 days and fluctuated around 3.7 x 10(7) trypanosomes/ml throughout the experimental period. The total CSF white cell counts increased from pre-infection means around 3 cells/micro l to a peak of 30 cells/micro l, 42 days post-infection (DPI). IL-10 was not detectable (<2 pg/ml) in serum prior to infection. IL-10 serum concentrations increased to 273 pg/ml 10 DPI coinciding with the first peak of parasitemia. Thereafter the levels declined to a mean value of 77 pg/ml 34 DPI followed by a significant rise to a second peak of 304 pg/ml (p<0.008) 42 DPI. There was no detectable IL-10 in CSF. IL-10 synthesis is thus stimulated both in the early and transitional stages of experimental trypanosomiasis. That IL-10 is produced in early stage disease is an interesting finding unlikely to be detected in humans where it is difficult to determine the exact time of infection. The IL-10 peak observed on day 42 of infection might indicate onset of parasite neuroinvasion coinciding with a peak in white blood cell counts in the blood and CSF.

The detection limits for estimates of infection intensity in schistosomiasis mansoni established by a study in non-human primates.

In human schistosomiasis mansoni, it is impossible to directly determine worm burden and hence infection intensity, so surrogates must be used. Studies on non-human primates revealed a linear relationship between worm burden and three surrogates, faecal egg output, circulating anodic and circulating cathodic antigens. By regression, the thresholds of detection were determined as 40, 24 and 47 worms, respectively. These observations provide a quantitative basis for the contention that low intensity infections in humans are being missed. The significance for estimates of disease prevalence, evaluation of the effects of chemotherapy and the implementation of vaccine trials is emphasised.

Previous or ongoing schistosome infections do not compromise the efficacy of the attenuated cercaria vaccine.

A current or previous schistosome infection might compromise the efficacy of a schistosome vaccine administered to humans. We have therefore investigated the influence of infection on vaccination, using the baboon as the model host and irradiated Schistosoma mansoni cercariae as the vaccine. Protection, determined from worm burdens in test and controls, was not diminished when vaccination was superimposed on a chronic infection, nor was it diminished when it followed a primary infection terminated by chemotherapy. Protection was also assessed indirectly based on fecal egg output and circulating antigen levels, as would be the case in human vaccine trials. In almost all instances, these methods overestimated protection, sometimes with discrepancies of >20%. The overwhelming immune response to egg deposition in infected animals made it difficult to discern a contribution from vaccination. Nevertheless, the well-documented immunomodulation of immune responses that follows egg deposition did not appear to impede the protective mechanisms elicited by vaccination with attenuated cercariae.

Parameters of the attenuated schistosome vaccine evaluated in the olive baboon.

Five exposures of baboons to the attenuated schistosome vaccine gave greater protection than three exposures, but this attenuation was not sustained when challenge was delayed. Within the scope of the data collected, fecal egg counts and circulating antigen levels did not accurately predict the observed worm burdens. Levels of immunoglobulin G at challenge correlated best with protection, but there was little evidence of a recall response.

Experimental infection of the olive baboon (Paplio anubis) with Plasmodium knowlesi: severe disease accompanied by cerebral involvement.

Experimental systems that model some of the complex interactions between parasite and host can be extremely valuable in identifying and developing new prophylactics and therapeutics against human diseases. Because primates have similar immune systems to humans, we have characterized a baboon model for understanding host response to Plasmodium knowlesi. Ten intact olive baboons (Papio anubis) of either sex were experimentally infected with P. knowlesi H strain erythrocytic parasites. The infection in these baboons was either acute or chronic. Animals with acute infection developed multiple system organ dysfunction and cerebral involvement. In chronically infected animals, only the spleen was moderately enlarged. The P. knowlesi parasitemia profile in baboons and rhesus monkeys was comparable. However, some clinical symptoms of the baboons and P. falciparum-infected humans were similar. These studies demonstrate for the first time that P. anubis is a suitable host for P. knowlesi for studying clinical symptoms and pathology.