Murine liver response to Allium sativum treatment during infection induced-trypanosomiasis

Hepatic injury induced by trypanosomiasis is one of the major health problems not only to human but also to wild and domestic animals. This study aimed to evaluate the hepatoprotective role of Allium sativum extract (ASE) against Trypanosoma evansi infection in mice. Animals were divided into 4 groups. Group I received only saline while group II received ASE (20 mg/Kg). Animals of group III and group IV were infected with T. evansi. The latter group was treated with ASE. The infrared spectroscopic analysis of A. sativum extract exhibited bands between 3700 cm⁻¹ and 599 cm⁻¹. On day 4 post T. evansi infection, ASE decreased the parasitemia by about 15 fold. Also, ASE regulated the number of erythrocytes and leucocytes and the hemoglobin content. In addition, the histopathological damage was reduced after treatment with ASE. Moreover, the oxidant and the antioxidant markers (glutathione, malondialdehyde and catalase) were regulated in the infected-treated animals. Collectively, the results proved the protective role of ASE against T. evansi infection in mice.     

Trypanosoma brucei brucei: A comparison of gene expression in the liver and spleen of infected mice utilizing cDNA microarray technology

Trypanosoma brucei brucei, the infectious agent of the disease known as Nagana, is a pathogenic trypanosome occurring in Africa, where it causes significant economic loss to domesticated livestock. Although many studies on the histopathology of organs of mice infected with T. b. brucei have been reported, little work has been done regarding gene expression in these organs in infected mice. In this paper, we describe the use of cDNA microarray to determine gene expression profiles in the liver and spleen of mice infected with T. b. brucei (STIB 920) at peak parasitaemia (12 days after infection). Our results showed that a total of 123 genes in the liver and 389 genes in the spleen were expressed differentially in T. b. brucei infected mice. In contrast, however, in an acute infection in mice caused by Trypanosoma brucei evansi, a species genetically related to T. b. brucei, 336 genes in the liver and 190 genes in the spleen were expressed, differentially, indicating that the liver of mice was more affected by the acute T. b. evansi infection whilst the spleen was more affected by the subacute T. b. brucei infection. Our results provide a number of possible reasons why mice infected with T. b. evansi die sooner than those infected with T. b. brucei: (1) mice infected with T. b. evansi may need more stress response proteins to help them pass through the infection and these are probably excessively consumed; (2) proliferating cell nuclear antigen was more down-regulated in the liver of mice infected with T. b. evansi, which indicated that the inhibition of proliferation of hepatocytes in mice infected with T. b. evansi might be more severe than that in T. b. brucei infection; and (3) more hepatocyte apoptosis occurred in the mice infected with T. b. evansi and this might be probably the most important reason why mice died sooner than those infected with T. b. brucei. Studies of the changes in the gene expression profile in the liver and spleen of mice infected with T. b. brucei may be helpful in understanding the mechanisms of pathogenesis in Nagana disease at the molecular level. By comparing the gene profiles of the liver and spleen of mice infected with T. b. brucei with T. b. evansi, we have identified a number of factors that could explain the differences in pathogenesis in mice infected with these two African trypanosomes.     

First identification of Trypanosoma vivax among camels (Camelus dromedarius) in Yazd,central Iran,jointly with Trypanosoma evansi

Trypanosomes are protozoan parasites of class Kinetoplastida. Trypanosoma vivax is one of the organisms that can cause Nagana and Trypanosoma evansi can cause Surra. In Africa, Trypanosoma vivax is mainly transmitted by Glossina spp. (tsetse fly) but it can be transmitted mechanically by other blood-feeding dipters. Trypanosoma evansi is transmitted mechanically and non-dependent to tsetse fly. In this research, T. vivax and T. evansi among camels (Camelus dromedarius) in Yazd, Iran were identified by microscopy and molecular examinations but the sensitivity of microscopy was lower than molecular examinations. Trypanosoma vivax and T. evansi were observed in 4 out of 134 blood film samples (2.98%). The prevalence of Trypanosoma spp. among 134 male camels (C. dromedarius) based on molecular examinations was 30.6% (22.76–38.44% with 95% confidence interval), 25 out of 134 (18.65%) had co-infection of T. evansi and T. vivax, and 16 out of 134 (11.94%) had an infection of T. vivax alone. We provided the first confirmation of infection with T. vivax among camels in Iran, and also in Asia, which has important implications on our knowledge of the occurrence and possible spread of this pathogen at the global level. Investigations in other species such as cattle and sheep are strongly recommended.     

Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice

Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse’s productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of highly parasitaemic mice that was detectable by PCR was 0.001 ng, using the ESAG6/7 and TBR1/2 primer, (b) using TBR1/2 primer for parasites purified could detect 0.000001 ng and (c) in the prepatent period PCR detect the presence of parasites earlier than parasitological techniques. Nevertheless, the percentage of detection for PCR varies depending on primer employed with 60% and 66% for ITS1 and 21/22-mer, and 80% for ESAG6/7 and TBR1/2. Consequently, TBR1/2 and ESAG6/7 were the best primers to monitor T. evansi infections in mice. For epidemiological application, such comparative evaluation should be made for detection of T. evansi in livestock such as horses.     

Investigations into human serum sensitivity expressed by stocks of Trypanosoma brucei evansi

Trypanosoma bruceievansi, a widely distributed species of trypanosome infecting different livestock species in many countries in Africa, Asia and South America, has recently been reported as a pathogen causing a case of human trypanosomiasis in India. To date, there is little information regarding the natural resistance of animal-infective stocks of T. b. evansi to normal human serum (NHS). In this study, we investigated the degree of sensitivity to NHS of 15 stocks of T. b. evansi from different geographical origins and found that 10 of the stocks were completely susceptible to the action of NHS; parasites disappeared from the blood of infected mice within a few hours and the mice remained free from infection for more than 1 month. The remaining five stocks were partially resistant to NHS; although parasites initially disappeared from the circulation more than 50% of the mice showed relapse infection 10-18 days later. Studies on one stock, T. b. evansi STIB 810, showed that the changes in parasitaemia in the infected mice were correlated with the amount of NHS inoculated (correlation factor −0.584 and P = 0.001). When this stock was passaged 25 times in mice in the presence of NHS it was found that the trypanosomes’ serum resistance increased compared with the parent stock from which they were derived; 40% of the passaged parasites survived after in vitro incubation with 50% NHS for 7 h, while only 1% of individual trypanosomes of the parent stock survived under the same conditions. These findings show, to our knowledge for the first time, that human serum sensitivity varies amongst stocks of T. b. evansi, that some of them naturally display resistance to NHS and that, furthermore, T. b. evansi serum resistance can be increased by sub-passage in the presence of NHS.     

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFα). TNFα has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFα. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 μg of antihuman TNFα blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFα treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFα production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.     

Protective effect of berberine chloride on Plasmodium chabaudi-induced hepatic tissue injury in mice

The present study aimed to investigate the protective role of berberine (BER) against Plasmodium chabaudi-induced infection in mice. Animals were divided into three groups. Group I served as a vehicle control. Group II and group III were infected with 1000 P. chabaudi infected erythrocytes. Group III was gavaged with 100 μl of 10 mg/kg berberine chloride for 10 days. All mice were sacrificed at day 10 post-infection. The percentage of parasitemia was significantly reduced more than 30%, after treatment of mice with BER. Infection caused marked hepatic injuries as indicated by histopathological alterations as evidenced by the presence of hepatic lobular inflammatory cellular infiltrations, dilated sinusoids, vacuolated hepatocytes, increased number of Kupffer cells and the malaria pigment, hemozoin. These changes in livers led to the increased histological score. Also, infection induced a significant increase in liver alanine aminotransferase and aspartate aminotransferase and a significant increase in the total leucocytic count. Moreover, mice became anemic as proved by the significant decrease in erythrocyte number and haemoglobin content. BER showed a significant protective potential by improving the above mentioned parameters. Based on these results, it is concluded that berberine could offer protection against hepatic tissue damage.     

Pre-treatment with curcumin modulates acetylcholinesterase activity and proinflammatory cytokines in rats infected with Trypanosoma evansi

The potent activity against Trypanosomes and health beneficial effects of curcumin (Cur) has been demonstrated in various experimental models. In this study, we evaluated the in vivo effect of Cur as trypanocide and as potential anti-inflammatory agent, through the evaluation of immunomodulatory mechanisms in rats infected with Trypanosoma evansi. Daily oral Cur was administered at doses of 0, 20 or 60 mg/kg as preventive treatment (30 and 15 days pre infection) and as treatment (post infection). The treatment of the groups continued until the day of euthanasia. Fifteen days after inoculation, parasitemia, plasma proinflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-6), anti-inflammatory cytokines (IL-10) and blood acetylcholinesterase activity (AChE) were analyzed. Pretreatment with Cur reduced parasitemia and lethality. Cur inhibited AChE activity and improved immunological response by cytokines proinflammatory, fundamental during T. evansi infection. We found that Cur is not so important as an antitrypanosomal activity but as immunomodulator agent. These findings reveal that the preventive use of Cur stimulates anti-inflammatory mechanisms, reducing an excessive inflammatory response.     

Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections

Background  

Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR).     

The effect of trans-ferulic acid and gamma-oryzanol on ethanol-induced liver injury in C57BL mouse

The effects of the oral administration of trans-ferulic acid and gamma-oryzanol (mixture of steryl ferulates) with ethanol (5.0 g per kg) for 30 days to c57BL mice on ethanol-induced liver injury were investigated. Preventions of ethanol-induced liver injury by trans-ferulic acid and gamma-oryzanol were reflected by markedly decreased serum activities of plasma aspartate aminotransferase, alanine aminotransferase and significant decreases in hepatic lipid hydroperoxide and TBARS levels. Furthermore, the trans-ferulic acid- and gamma-oryzanol-treated mice recovered ethanol-induced decrease in hepatic glutathione level together with enhancing superoxide dismutase activity. These results demonstrate that both trans-ferulic acid and gamma-oryzanol exert a protective action on liver injury induced by chronic ethanol ingestion.     

Influence of treatment with 3′-deoxyadenosine associated deoxycoformycin on hematological parameters and activity of adenosine deaminase in infected mice with Trypanosoma evansi

This study aimed to verify the effect of 3′-deoxyadenosine and deoxycoformycin on hematologic parameters and adenosine deaminase (ADA) activity in plasma and brain of mice infected with Trypanosoma evansi. Seventy animals were divided into seven groups, which were divided into two subgroups each for sampling on days 4 and 8 post-infection (PI). The groups were composed of three uninfected groups (A–C), namely, not-treated (A), treated with 3′-deoxyadenosine (B), and treated with deoxycoformycin (C) and four infected groups, mice with T. evansi (D–G), namely, not-treated (D), treated with 3′-deoxyadenosine (E), treated with deoxycoformycin (F), and treated with a combination 3′-deoxyadenosine and deoxycoformycin (G). Hematological parameters and ADA activity were evaluated in plasma and brain. Animals in groups B and C exhibited a reduction in the levels of plasma total protein compared group A. Animals in groups D and F showed changes in the hematological parameters. The ADA activity significantly reduced in the animals of groups C, D, F and G. Mice in the group E presented increased ADA activity in plasma. Therefore, we conclude that the treatment interferes significantly in the hematologic parameters in mice infected with T. evansi. On the other hand, when the ADA inhibitor was used we observed a significant decrease in the values of hematocrit, total erythrocytes, and hemoglobin concentration. The deoxycoformycin was able to inhibit the ADA activity of parasite thus it may be one of the mechanisms of efficacy of this treatment.     

Vitamin B12 blocked Trypanosoma brucei rhodesiense-driven disruption of the blood brain barrier,and normalized nitric oxide and malondialdehyde levels in a mouse model

Infection with Trypanosoma brucei rhodesiense (T.b.r) causes acute Human African Trypanosomiasis (HAT) in Africa. This study determined the effect of vitamin B12 on T.b.r -driven pathological events in a mouse model. Mice were randomly assigned into four groups; group one was the control. Group two was infected with T.b.r; group three was supplemented with 8 mg/kg vitamin B12 for two weeks; before infection with T.b.r. For group four, administration of vitamin B12 was started from the 4th days post-infection with T.b.r. At 40 days post-infection, the mice were sacrificed to obtain blood, tissues, and organs for various analyses. The results showed that vitamin B12 administration enhanced the survival rate of T.b.r infected mice, and prevented T.b.r-induced disruption of the blood-brain barrier and decline in neurological performance. Notably, T.b.r-induced hematological alteration leading to anaemia, leukocytosis and dyslipidemia was alleviated by vitamin B12. T.b.r-induced elevation of the liver alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin as well as the kidney damage markers urea, uric acid and creatinine were attenuated by vitamin B12. Vitamin B12 blocked T.b.r-driven rise in TNF-α and IFN-γ, nitric oxide and malondialdehyde. T.b.r-induced depletion of GSH levels were attenuated in the presence of vitamin B12 in the brain, spleen and liver tissues; a clear indication of the antioxidant activity of vitamin B12. In conclusion, treatment with vitamin B12 potentially protects against various pathological events associated with severe late-stage HAT and presents a great opportunity for further scrutiny to develop an adjunct therapy for severe late-stage HAT.     

Effects of anthraquinone extract from Rheum officinale Bail on the physiological responses and HSP70 gene expression of Megalobrama amblycephala under Aeromonas hydrophila infection

We evaluated the effect of dietary supplementation with anthraquinone extract (from Rheum officinale Bail) on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into two groups: a control group (fed a standard diet) and a treatment group (standard diet supplemented with 0.1% anthraquinone extract) and fed for 10 weeks. We then challenged the fish with A. hydrophila and recorded mortality and changes in serum cortisol, lysozyme, alkaline phosphatase (ALP), total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and the relative expression of heat shock protein 70 (HSP70) mRNA for a period of 5 d. Supplementation with 0.1% anthraquinone extract significantly increased serum lysozyme activity before infection, serum ALP activity at 24 h after infection, serum total protein concentration 12 h after infection, hepatic CAT activity 12 h after infection, hepatic SOD activity before infection, and the relative expression of hepatic HSP70 mRNA both before infection and 6 h after infection. In addition, the supplemented group had decreased levels of serum cortisol 6 h after infection, serum AST and ALT activities 12 h after infection, and hepatic MDA content 12 h after infection. Mortality was significantly lower in the treatment group (86.67%) than the control (100%). Our results suggest that ingestion of a basal diet supplemented with 0.1% anthraquinone extract from R. officinale Bail can enhance resistance against pathogenic infections in M. amblycephala.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

The immunological response of CBA mice to Trypanosoma musculi: mechanisms of protective immunity

CBA mice which had recovered from infection with Trypanosoma musculi were immune to challenge with all strains of the homologous species that were tested but were still full susceptible to challenge with T. cruzi, T. brucei or T. evansi. A heavy challenge inoculum of T. musculi was cleared rapidly from the blood of mice which had recently recovered from infection but, in mice which had recovered 11 months earlier, the parasitaemia changed very little for 3–4 days but then fell abruptly within a few hours. Immunization with a parasite extract in multiple emulsion conferred a strong though not complete protection against homologous challenge.Serum from mice which had recovered from infection had a marked neutralizing effect in vitro on the infectivity of the homologous parasites although the numbers of live organisms were not reduced during the period of in vitro incubation. The test did not reveal antigenic differences among three isolates of the parasite.A summary is given of the sequence of events that is thought to make up the immune response of mice to T. musculi.     

Trypanosoma evansi kDNA minicircle found in the Venezuelan nectar-feeding bat Leptonycteris curasoae (Glossophaginae), supports the hypothesis of multiple origins of that parasite in South America

Trypanosoma evansi is a mammal generalist protozoon which causes negative effects on health and productivity in bovine and equine herds in South America, Europe, Asia and Africa. By molecular methods, we screened the presence of that parasite together with other trypanosome species in 105 bats of 10 species collected in arid zones of northern Venezuela. The first molecular approach was fluorescent fragment length barcoding (FFLB), which relies on amplification of relative small regions of rRNA genes (four loci) and fluorescence detection. By FFLB, 17 samples showed patterns of possible trypanosomatid infections. These samples were used to test presence of trypanosomes by PCR using the following DNA markers: V7–V8 SSU rRNA, gGAPDH and kDNA minicircle regions. Only in one individual of the nectar-feeding bat, Leptonycteris curasoae, we were able to amplify 1000 bp of the trypanosome kDNA minicircle. That PCR product was sequenced and the parasite species was determined by NCBI-BLAST and phylogenetic analysis. Both analyses showed that the minicircle sequence corresponds to Trypanosoma evansi. The phylogenetic analysis of the sequence obtained in this study clustered with a T. evansi sequence obtained in a Venezuelan capybara, Hydrochoerus hydrochaeris, and distant of others two T. evansi sequences obtained in a Colombian capybara and horse. This result supports the hypothesis of multiple origins of T. evansi in South America.     

Immunological studies of the mechanism of anaemia in experimental Trypanosama evansi infection in rats

Pathological changes in the blood of rats acutely infected with Trypanosoma evansi and the probable mechanism of the accompanying anaemia, were investigated. A severe anaemia, together with rreticulocytosis and hepato-splenomegaly, were regularly observed. Histological examination of the liver, spleen and bone-marrow confirmed the increasedin erythropoietic activity that the observed anaemia was due to increasedextravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity. All the infected rats showed significant immune responses to the infecting trypanosome peak agglutinin titres occurring 10–12 days after injection, coincidentally with maximun destruction of erythrocytes. Serological examination of sera and erythrocytes from all infected and control rats did not reveal the presence of either circulating or adsorbed erythrocyte auto--antibodies. Furthermore, there was no in vivo trypanosomal antigen coating of the erythrocytes from either infected or multiple antigen-injected rats. Repeated intraveoous injections into rats of more than 100 μg per g body weight of soluble T. evansi antigen resulted in moderately severe, probably antibody-mediated, haemolytic anaemia. It is considered that an immunologically-mediated mechanism may be responsible for the development of the anaemia accompanying T. evansi infection.     

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.     

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 min at 63 °C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T_m) of ∼89 °C. The assay analytical sensitivity is ∼0.1 tryps/ml while that of classical PCR test targeting the same gene is ∼10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.