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Haemonchus contortus is a highly pathogenic and prevalent helminth that causes many deaths in sheep herds. Anthelmintics are usually employed to overcome this issue; however, they do not guarantee immediate and lasting efficacy because of the occurrence of drug-resistant parasites. Among substances that are used in scientific studies for parasitic control, essential oils are known to have different pharmacological properties. However, they demonstrate instability owing to several factors, and therefore, nanoemulsification is considered an alternative to control the instability and degradability of these compounds. The objective of this study was to evaluate the cytotoxicity of nanoemulsions containing essential oil of Eucalyptus globulus against the blood of healthy sheep and to verify their activity against the parasite H. contortus in sheep. The results presented adequate nanotechnological characteristics (diameter 72 nm, PDI 0.2, zeta -11 mV, and acidic pH) and adequate morphology. Further, the corona effect and cytotoxic profiles of the free oil and nanoemulsion against blood cells from healthy sheep were evaluated. The tests results did not present a toxicity profile. For evaluating efficacy, we observed an important anthelmintic action of the nanoemulsion containing oil in comparison to the free oil; the results demonstrate a potential role of the nanoemulsion in the inhibition of egg hatchability and the development of larvae L1 to L3 (infective stage). Based on these results, we developed an important and potential anthelmintic alternative for the control of the parasite H. contortus.

Infection is one of the main complications that hinder wound healing. Currently, antibiotic-resistant bacteria, such as Methicilin-resistant Staphylococcus aureus (MRSA), are a concern worldwide for both humans and animals. Maggot therapy is re-emerging as an alternative to intractable wounds and may be an option to the traditional antibiotic treatment. Although the species of choice is Lucilia sericata, reports of clinical use have led us to evaluate the efficacy and safety of using Lucilia cuprina larvae on induced infected wounds in Wistar rats. In short, 32 male Wistar rats were divided into 4 groups: Group I - saline solution treated; Group II - antibiotic-treated; Group III - treated with larval debridement, and Group IV - without wound and treatment. Skin wounds were induced in groups I, II and III. All treatments were performed once and held for 48 h. Clinical, microbiological, histopathological, hematological, and biochemical analyses were done. Significant wound area contraction was found (95%) in group III on day 9 compared to day 15 in group II. Complete elimination (0.0 ± 0.0 CFU/mL) of bioburden was achieved after the second treatment (day 6) in both the II and III groups, compared to an increase in Group I (6.51 ± 0.37 CFU/mL). A cleaner wound was also observed in the histopathological evaluation of group III, with adequate collagen formation and re-epithelialization on day 15. Furthermore, larvae increased blood platelet levels after the first treatment. L. cuprina larvae have proven safe and effective in accelerating wound treatment and eliminating MRSA.

Chagas disease, caused by Trypanosoma cruzi, is a major public health problem and is described as one of the most neglected diseases worldwide. It affects about 6-7 million people. Currently, only two drugs are available for the treatment of this disease: nifurtimox and benznidazole. However, both drugs are highly toxic and have several side effects, which lead many patients to discontinue treatment. Moreover, these compounds show a significant curative efficacy only in the acute phase of the disease. Therefore, searching for new drugs is necessary. The objective of this study was to evaluate the in vitro and in vivo activity of a benzofuroxan derivative (EA2) against T. cruzi, and to evaluate the hematological and biochemical changes induced by its treatment in animals infected with T. cruzi. The results were then compared with those of healthy controls. In vitro testing was first performed with T. cruzi epimastigote forms. In this experiment, EA2 was diluted at three different concentrations (0.25, 0.50, and 1%). In vitro evaluation of the trypanocidal activity was performed 24, 48, and 72 h after incubation. In vivo assays were performed using three different doses (10, 5, and 2,5 mg/kg). Mice were divided into 10 groups (five animals/group), wherein four groups comprised non-infected animals (A, G, H, I) and six groups comprised infected animals (B, C, D E, F, J). Groups B and J represented the negative and positive controls, respectively. Groups G, H, and I were used to confirm that EA2 was not toxic to non-infected animals. Parasitemia was measured in infected animals and the hematological and biochemical profiles (urea, creatinine, albumin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) were evaluated in all animals. EA2 demonstrated in vitro trypanocidal activity at all concentrations tested. Although it did not demonstrate a curative effect in vivo, EA2 was able to retard the onset of parasitemia, and significantly reduced the parasite count in groups D and E (treated with 5 and 2.5 mg/kg, respectively). EA2 did not induce changes in hematological and biochemical parameters in non-infected animals, demonstrating that it is not toxic. However, further assessments should aim to confirm the safety of EA2 since this was the first in vitro and in vivo study conducted with this molecule.

The insecticidal activity of Myristica fragrans (Houtt) essential seed oil, (Nutmeg) was evaluated against Musca domestica (Linnaeus) and Chrysomya albiceps (Wiedemann); both important infectious pathogenic disease vectors. The oil was extracted by distillation, and 21 components were identified during chemical analysis; principally β-pinene (26%), α-pinene (10.5%), Sabinene (9.1%) and γ-terpinen (8.5%). Insecticidal properties were identified through larvicide and adulticide tests. Using the immersion method, the oil at 5% was found to be very effective (90 ± 1%) against M. domestica larvae. The results for adulticide activity varied by fly species, dosage, time, and method of exposure. Topical application (on the insect thorax) was more toxic to C. albiceps, where the lethal concentration at 50% (LC50) was 2.02 ± 0.56, and 8.57 ± 2.41 for the common flies. When the insects were exposed to oil impregnated paper, the results were similar for M. domestica and C. albiceps adults with respective LC50 values of 2.74 ± 0.24, and 3.65 ± 0.48. Thus, the results demonstrated that M. fragrans oil presents insecticidal activity and can be used for control of Musca domestica and Chrysomya albiceps.

The aim of this study was to evaluate the activity of purinergic enzymes in lymphocytes and cardiac tissue of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. On day 12 post-infection (PI), the animals were anesthetized and after euthanized, and samples were collected for analyses. Infected mice showed reduction in erythrocyte counts, hematocrit and hemoglobin concentration, as well as reduced number of total leukocytes in consequence of neutrophilia (P 0.01). The number of monocytes increased in infected mice (P 0.001), however the number of lymphocytes and eosinophils did not differ between groups (P 0.05). The E-NTPDase (ATP and ADP substrate) and E-ADA activities in lymphocytes increased significantly in mice infected by T. cruzi (P 0.01). In the heart, multiple pseudocysts containing amastigotes within cardiomyocytes were observed, as well as focally extensive severe necrosis associated with diffuse moderate to severe inflammatory infiltrate of lymphocytes. Although, the NTPDase activity (ATP and ADP substrate) in the cardiac homogenate did not differ between groups, a reduction on 5'-nucleotidase activity (P 0.001) and an increase in the ADA activity in infected animals (P 0.05) were observed. Thus, animals infected by T. cruzi experienced the disease, i.e., showed anemia, leucopenia, and heart lesions. Associated with this, purinergic enzymes showed altered activities, which might be related to the modulation of the inflammatory response.

This study aimed to evaluate the susceptibility in vitro and in vivo of Trypanosoma evansi to terpinen-4-ol, γ-terpinene and α-terpinene, the three main compounds of tea tree oil (Melaleuca alternifolia) with known efficacy in the treatment of trypanosomosis. In vitro, a trypanocidal effect of terpinen-4-ol, γ-terpinene, and α-terpinene was observed when used alone or associated at 0.5, 1 and 2% concentrations i.e., the α-terpinene showed a faster trypanocidal effect when compared to chemotherapy (diminazene aceturate - D.A.). In vivo studies were performed in two experiments: I and II where experiment I used T. evansi infected mice treated with terpinen-4-ol, γ-terpinene and α-terpinene alone (at a dose of 1.0 mL kg(-1)) or associated (two compounds, dose of 0.5 mL kg(-1) of each compound; tree compounds, dose of 0.335 mL kg(-1) of each compound); Treatment with α-terpinene was able to extend animal longevity, but showed no curative efficacy. In experiment II, T. evansi infected mice were treated with D.A. associate with α-terpinene, where a curative efficacy of 57.14% was found, a much better result when D.A. was used alone (14.28%). In summary, α-terpinene associated with D.A. can be used as an alternative treatment for T. evansi infection. The compound α-terpinene from M. alternifolia essential oil is the one responsible for the trypanocidal effect, a fact confirmed by in vitro results and the increased longevity observed on treated mice.

The aim of this study was to evaluate in vitro the efficacy of cordycepin and pentostatin (alone or combined) against Trypanosoma cruzi, as well as the therapeutic efficiency of protocols of cordycepin and pentostatin combinations in mice experimentally infected with T. cruzi. In vitro, the cordycepin (3′-deoxyadenosine) and pentostatin (deoxycoformycin) exerted potent trypanocidal effect against T. cruzi (Colombian strain), similarly to benznidazole, which is the reference drug. For epimastigotes, the lethal dose of cordycepin capable of killing 50% (LD50) and 20% (LD20) of the parasites was 0.072 and 0.031 mg/mL, respectively and for trypomastigotes was 0.047 and 0.015 mg/mL, respectively. The combined use of cordycepin and pentostatin resulted in a LD50 and LD20 for epimastigotes of 0.068 and 0.027 mg/mL, respectively, as well as 0.056 and 0.018 mg/mL for trypomastigotes, respectively. In vivo, the combined use of cordycepin and pentostatin did not show the expected curative effect, however it was able to control the parasitema in the peak period. In summary, the combination of cordycepin and pentostatin showed no curative effect in mice infected by T. cruzi, despite the in vitro reduction of epimastigotes and trypomastigotes.

This study aimed to verify the effect of the treatment with A. satureioides essential oil (free and nanoencapsulated forms) and diminazene aceturate on hematological and biochemical variables in rats infected by Trypanosoma evansi. The 56 rats were divided into seven groups with eight rats each. Groups A, C and D were composed by uninfected animals, and groups B, E, F and G were formed by infected rats with T. evansi. Rats from groups A and B were used as negative and positive control, respectively. Rats from the groups C and E were treated with A. satureioides essential oil, and groups D and F were treated with A. satureioides nanoencapsulated essential oil. Groups C, D, E and F received one dose of oil (1.5 mL kg(-1)) during five consecutive days orally. Group G was treated with diminazene aceturate (D.A.) in therapeutic dose (3.5 mg kg(-1)) in an only dose. The blood samples were collected on day 5 PI for analyses of hematological (erythrocytes and leukocytes count, hemoglobin concentration, hematocrit, mean corpuscular and mean corpuscular hemoglobin concentration) and biochemical (glucose, triglycerides, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, urea and creatinine) variables. A. satureioides administered was able to maintain low parasitemia, mainly the nanoencapsulated form, on 5 days post infection. On the infected animals with T. evansi treated with A. satureioides essential oil (free and nanocapsules) the number of total leucocytes, lymphocytes and monocytes present was similar to uninfected rats, and different from infected and not-treated animals (leukocytosis). Treatment with A. satureioides in free form elevated levels of ALT and AST, demonstrating liver damage; however, treatment with nanoencapsulated form did not cause elevation of these enzymes. Finally, treatments inhibited the increase in creatinine levels caused by infection for T. evansi. In summary, the nanoencapsulated form showed better activity on the trypanosome; it did not cause liver toxicity and prevented renal damage.

The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes of lambs experimentally infected by Haemonchus contortus. A total of 14 healthy lambs were used, divided into two groups of seven animals each. Group A (negative control) represented the uninfected animals, and Group B (positive control) was formed by animals infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the white blood cells (WBC) count, as well as the evaluation of AChE activity in lymphocytes. Parasitological stool exam (eggs per gram of feces - EPG) was performed on the same days to follow up the evolution of the infection. On day 15 PI it was verified negative EPG; however, on days 45 and 75 PI it was observed positive EPG only in the animals of group B. In the three evaluated periods was observed a lower number of leukocytes, associated with decreased lymphocytes and neutrophils in lambs infected by this gastrointestinal nematodes. Lambs infected with H. contortus showed significant (P0.01) lower AChE activity in lymphocytes compared uninfected. Statistically, there was a positive correlation (P0.05) between AChE activity in lymphocytes and number of lymphocytes (r=0.69). The lymphocytes are cells with direct participation in the cholinergic system; therefore, based on these results, it can be concluded that the experimental infection with H. contortus influences the number of lymphocytes, and consequently the AChE activity in these cells.

The aims of this study were to evaluate if the use of copper oxide wire particles, isolated or in association with closantel, in lambs infected with Haemonchus contortus enhances the anthelmintic efficacy of closantel, as well as to evaluate the effects of treatment in hepatic energy metabolism, inflammatory markers and hematological and biochemical tests. The lambs were randomly divided into five groups (6 animals each), as follows: uninfected animals (Control); animals infected with H. contortus (HC); infected and treated with closantel (HC + CL); infected and treated with copper oxide wire particles (HC + Cu); and infected and treated with closantel plus copper oxide wire particles (HC + CL + Cu). The animals of infected groups were infected orally with H. contortus (5,000 L3 -larvae) and on day 14 post infection (p.i) the treatments were initiated. The egg per gram of feces (EPG), butyrylcholinesterase (BuChE), myeloperoxidase (MPO), adenylate kinase (AK) and pyruvate kinase (PK) activities and hematological and biochemical tests were evaluated. Treatments with copper oxide (isolated and associated) were able to reduce the EPG count on days 28, 35, 42 and 49 p.i when compared to HC group, while closantel was able to reduce EPG only from day 35 p.i. Moreover, treatment with closantel (isolated or associated) was able to prevent the inhibition of hepatic AK and PK activities caused by H. contortus infection, which may contribute to efficient intracellular energetic communication in order to maintain the balance between cellular ATP consumption and production. Butyrylcholinesterase and MPO activities were higher in infected lambs compared to uninfected, while treated groups showed lower enzymatic activity compared to the group HC. The use of all therapeutic protocols was able to reduce the EPG count. Based on these evidences, the use of copper oxide plus closantel may be considered an alternative to treat lambs infected by H. contortus.

The goal of this study was to evaluate reproductive hormones in sera samples of female rats experimentally infected by Trypanosoma evansi during different phases of the estrous cycle. For that, 64 animals were divided into two groups: 24 rats for the control group (uninfected), and 40 animals were infected by T. evansi. These groups were divided into subgroups according to the time of infection (days 5 and 15 post-infection; PI) and the phase of the estrous cycle (proestrus, estrus, metestrus and diestrus). Serum was collected at days 5 and 15 PI and the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone and estradiol were assessed by enzyme immunoassay technique. The concentration of nitrite/nitrate (NOx), advanced oxidation protein products (AOPP), and thiobarbituric acid reactive substances (TBARS) were measured in ovaries and uteruses in these same periods. Infected females showed significant decrease (P

This study aimed to evaluate the susceptibility of Brazilian isolates of Trypanosoma evansi to suramin sodium. For this purpose, three isolates of T. evansi (LPV-2005, LPV-2009 and LPV-2010) and seventy mice were used, with the animals divided in 10 groups (A, B, C, D, E, F, G, H, I and J) with seven animals each group. Mice of groups A, B, and C were infected with LPV-2005; Groups D, E and F with LPV-2009 and the groups G, H and I with LPV-2010. The group J was composed by healthy mice or uninfected. The parasitemia was monitored daily through blood smear, and the treatment of all groups was performed three days post-infection (PI), when all mice showed increased parasitemia. Groups A, D and G represented the positives controls, while groups B, E and H received a single dose of suramin sodium at 10 mg kg −1 intramuscularly. Groups C, F and I were treated with three doses of suramin sodium at 10 mg kg −1 , respecting an interval of 24 h between each dose. Negative blood smears from all animals were obtained 24 h after treatment (AT), status maintained until the end of the experiment (50 days PI). The specific PCR for T. evansi was carried out from blood, showing negative results AT. Therefore, this study showed that a single dose of suramin sodium at 10 mg kg −1 has the same efficacy of three doses, as recommended by the therapeutic literature. Furthermore, we observed that Brazilian isolates did not show resistance to the drug.

The aim of this study was to evaluate the in vitro and in vivo susceptibility of Trypanosoma evansi to α-Bisabolol and solid lipid nanoparticles containing α-Bisabolol (SLN-B). In vitro, a trypanocidal effect of α-Bisabolol and SLN-B was observed when used at 0.5, 1 and 2% concentrations, i.e., the concentrations of 1 and 2% showed a faster trypanocidal effect when compared to chemotherapy (diminazene aceturate - D.A.). T. evansi infected mice were treated with α-Bisabolol and SLN-B at a dose of 1.0 mL kg-1 during seven days via oral gavage. In vivo, treatment with SLN-B, D.A. and D.A. associated with SLN-B were able to increase (p

The aims of this study were to develop nerolidol-loaded nanospheres, and to evaluate their efficacy in vitro and in vivo against Trypanosoma evansi, as well as to determine their physicochemical properties, morphology, and any possible side effect in vitro against peripheral blood mononuclear cell (PBMC). The nanospheres showed an adequate particle size (149.5 nm), narrow particle distribution (0.117), negative zeta potential (-12.8 mV), and pH of 6.84, such as observed by transmission electron microscopy. In vitro, a trypanocidal effect of nerolidol and nanospheres containing nerolidol was observed at 0.5, 1.0, and 2.0%, i.e., both treatments showed a faster trypanocidal effect compared to chemotherapy (diminazene aceturate - D.A.). T. evansi infected mice were used to evaluate the effects of nerolidol-loaded nanospheres regarding pre-patent period, longevity, and therapeutic efficacy. Oral administration of nerolidol-loaded nanospheres at 1.0 mL/kg/day during 10 days increased mice survival (66.66%) compared to 0% and 33.33% of mice survival when treated with nerolidol in its free form and D.A., respectively. Cytotoxic study indicated that both treatments showed no side effects in vitro against PBMC, an important marker used in toxicological surveys. Therefore, nanoencapsulation increased the therapeutic efficacy of nerolidol against T. evansi, and can be used as an alternative treatment for T. evansi infection.

The aim of this study was to evaluate the effects of a treatment using injectable zinc and copper in rats infected with Trypanosoma evansi. 48 rats were divided into eight groups of six animals each. Group A was composed of uninfected animals. Animals from groups B-H were inoculated at the 5th day of experiment with 1.2×10(6) trypanosomes. Group B was used as a positive control. The infected groups received prophylactic (C, D and E) and therapeutic (F, G and H) treatments with the zinc and copper, both at a dose of 5 mg kg(-1). The effectiveness of treatment was confirmed by negative blood smears and Polymerase Chain Reaction (PCR) at the end of study. All treated animals had their prepatent period and survival prolonged when compared with control group (group B). Treatment efficacy was 17% (C: zinc), 33% (D: copper), 50% (E: zinc+copper), 0% (F: zinc), 50% (G: copper) and 50% (H: zinc+copper). Thus, we can conclude that treatment with zinc and copper are capable of controlling and/or curing T. evansi infection in rats, delaying the parasitemia and prolonging their survival.

The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by Trypanosoma evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2mL of blood containing 10(6) trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.

The study was undertaken to evaluate changes in the activity of adenosine deaminase (ADA) in brains of rats infected by Trypanosoma evansi. Each rat was intraperitoneally infected with 10(6) trypomastigotes either suspended in fresh (group A; n = 13) and cryopreserved blood (group B; n = 13). Thirteen animals were used as control (group C). ADA activity was estimated in the cerebellum, cerebral cortex, striatum and hippocampus. No differences (P0.05) in ADA activity were observed in the cerebellum between infected and non-infected animals. Significant (P0.05) reductions in ADA activity occurred in cerebral cortex in acutely (day 4 post-infection; PI) and chronically (day 20 PI) infected rats. ADA activity was significantly (P0.05) decreased in the hippocampus in acutely infected rats, but significantly (P0.05) increased in the chronically infected rats. Significant (P0.05) reductions in ADA activity occurred in the striatum of chronically infected rats. Parasites could be found in peripheral blood and brain tissue through microscopic examination and PCR assay, respectively, in acutely and chronically infected rats. The reduction of ADA activity in the brain was associated with high levels of parasitemia and anemia in acute infections. Alterations in ADA activity of the brain in T. evansi-infected rats may have implications for pathogenesis of the disease.

The aim of this study was to evaluate cholinesterase activity during the early acute phase of Trypanosoma evansi infection in rats. Fifteen male Wistar rats were randomly distributed into three groups ( n = 5 animals per group): two trypanosome-infected groups (T3 and T5) and uninfected controls (C). The animals were inoculated intraperitoneally with 10 6 trypanosomes. The blood was collected by cardiac puncture on the 3rd (T3) or 5th day post-infection (T5 and C). Cerebrum and cerebellum were removed for the evaluation of acetylcholinesterase (AChE) activity. AChE activity was also evaluated in whole blood and butyrylcholinesterase activity (BUChE) in plasma samples. Parasitemia were progressive increase and parasites were observed in the peripheral blood of all infected animals one day post-inoculation. AChE activity was not altered in cerebrum and cerebellum tissues. AChE activity in blood significantly decreased in the T3 and T5 groups (26.63 and 25.86 mU/l mol Hb) compared with the control (37.84 mU/l mol Hb). In addition BUChE activity in plasma was lower in the T3 (7.01 μmol BTC hydrolyzed/h/mL) than the T5 and C groups (9.84 and 12.00 μmol BTC hydrolyzed/h/mL). This study therefore, shows that reductions in the activity of cholinesterase occur in acute infection by T. evansi in rats and this demonstrates an important change occurring in animals infected by the protozoan and may indicate a potential role the enzymes play in the mechanism of disease.

The aim of this study was to evaluate the concentrations of copper, iron and zinc in blood serum of cats experimentally infected with Trypanosoma evansi. Animals were divided into two groups: control and infected with T. evansi. The animals were infected with 10(8) trypomastigotes each and parasitemia was estimated daily for 56 days by microscopic examination of smears. Hematological and biochemical parameters were evaluated for monitoring of the disease. Serum metal levels were determined in blood samples collected at days 7, 28 and 56 of the experiment. Inductively coupled plasma optical emission spectrometry was used to measure the levels of copper, iron and zinc. Significant differences were observed among groups (P0.05). Increased levels of copper and decreased iron and zinc levels were observed. A decrease in the number of red blood cells was also observed 7 days after inoculation. Biochemical parameters were not altered. Therefore, the infection by T. evansi might alter the serum metal levels, causing metabolic disturbances in cats.

This study aimed at evaluating hemogram and erythropoietic changes in cats experimentally infected with Trypanosoma evansi. Thirteen adult female non-breeding Felix catus were separated into two groups: seven animals were infected with 10(8) trypomastigotes each, and six animals were used as negative controls. Animals were kept in air-conditioned rooms and blood smears were performed daily for 49 days. Blood samples were collected from the jugular vein at days 0, 7, 21, 35 and 49 and stored in blood-collecting tubes containing anticoagulant. Bone marrow was collected from the proximal epiphysis of the right femur at days 14 and 42 post-inoculation (PI). Total erythrocyte count, hematocrit and hemoglobin showed statistical differences among groups from the seventh day PI onwards (P0.05). The mean corpuscular volume and the mean corpuscular hemoglobin concentration remained normal, characterizing a normocytic-normochromic anemia. Reticulocyte count increased in the infected group from the 21st day onwards, but remained near normal values suggesting a mild regenerative anemia. Moreover, the myeloid:erythroid ratio significantly reduced at day 42 PI, evidencing a bone marrow hematopoietic response. Based on these results we conclude that cats infected with T. evansi have normocytic, normochromic, regenerative anemia.

Gastrointestinal parasites are one of the biggest health problems faced in sheep, mainly due to their pathogenicity and resistance to drugs used to control these parasites. Thus, the following study aimed to assess the anthelmintic efficacy of Melaleuca alternifolia against Haemonchus contortus in gerbils (Meriones unguiculatus) experimentally infected. Three treatments were tested: M. alternifolia essential oil, popularly known as tea tree oil (TTO), a solid lipid nanocarrier made with essential oil of Melaleuca (nanoTTO), and terpinen-4-ol (terp-4-ol). In vivo studies were performed by determining the mean worm burden of H. contortus in gerbils TTO (0.75 mL/kg); nanoTTO (0.5 mL/kg) and terp-4-ol (0.5 mL l/kg) were able to reduce 46.36%; 48.64%, and 43.18% worm burden, respectively. H. contortus increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as demonstrated by liver injury. It was found that the TTO, nanoTTO, and terp-4-ol were not toxic to liver and kidneys since hepatic and renal functions were not affected. Moreover, terp-4-ol was able to prevent increased levels of seric AST and ALT in infected animals, indicating a hepatoprotective effect. Thus, our results indicate that TTO, nanoTTO, and terp-4-ol are safe and efficient against H. contortus infection in gerbils, and possibly the terp-4-ol may be considered the compound present in the Melaleuca alternifolia responsible for parasitic action against H. contortus.

Trypanosoma evansi is an important pathogen that causes changes in nitric oxide (NO) levels and antioxidant enzymes, as well as oxidative stress. The present study evaluated the in vivo effect of T. evansi infection on frequency and index of DNA damage in liver, heart, spleen and total blood of rats. Twenty rats were assigned into two groups with ten rats each, being subdivided into four subgroups (A1 and A2, 5 animals/group; and B1 and B2, 5 animals/group). Rats in the subgroups A1 and A2 were used as control (uninfected) and animals in the subgroups B1 and B2 were inoculated with T. evansi (infected). NO in serum and the comet assay were used to measure DNA damage index (DI) and damage frequency (DF) in liver, heart, spleen and total blood of infected rats. Increased NO levels on days 3 and 9 post-infection (PI) was observed (P

Biochemical and molecular research on parasites has increased considerably in trypanosomes in the recent years. Many of them have the purpose of identify areas, proteins and structures of the parasite which are vulnerable and could be used in therapy against the protozoan. Based on this hypothesis this study aimed to detect biochemically the enzyme adenosine deaminase (ADA) in Trypanosoma evansi, and to adapt an assay to the measurement of its activity in trypomastigotes. Firstly, the parasites were separated from the blood of mice experimentally infected with a DEAE-cellulose column. The ADA activity in trypomastigotes was evaluated at concentrations of 0.1, 0.2, 0.5, 0.6 and 0.8 mg of protein by spectrophotometry. ADA activity was observed in the parasites at all concentrations tested and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.24 U/L in the lowest and highest concentration of protein, respectively. Therefore, it is possible to detect biochemically ADA in T. evansi, an enzyme that may be associated with vital functions of the parasite, similar to what occurs in mammals. This knowledge may be useful in the association of the chemotherapic treatment with specific inhibitors of the enzyme, in future studies.

The aim of this study was to investigate the behavioral assessment and activities of important enzymes involved in the phosphoryl transfer network in rat brains that were experimentally infected with Trypanosoma evansi. Behavioral assessment (cognitive performance), pro-inflammatory cytokines in serum and activities of adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in brain were evaluated at 5 and 15 days post-infection (PI). Here we demonstrate a cognitive impairment in the rats infected with T. evansi. At 5 and 15 days PI, a memory deficit and a depressant activity were demonstrated by an inhibition avoidance test and increase in the immobility time in a tail suspension test, respectively. On day 5 PI, a decrease in the CK activity and an increase in the AK activity were observed. On day 15 PI, an increase in the CK activity and a decrease in the AK activity were observed. Considering the importance of energy metabolism for brain functioning, it is possible that the changes in the activity of enzymes involved in the cerebral phosphotransfer network and an increase in the proinflammatory cytokines (TNF and IFN) may be involved at least in part in the cognitive impairment in infected rats with T. evansi.

The aim of this study was to assess the effects of iron supplementation on oxidative stress and on the activity of the adenosine deaminase (ADA) in rats experimentally infected by Trypanosoma evansi. For this purpose, 20 rats were divided into four experimental groups with five animals each as follows: groups A and B were composed by healthy animals, while animals from groups C and D were infected by T. evansi. Additionally, groups B and D received two subcutaneous doses of iron (60 mg kg-1) within an interval of 5 days. Blood samples were drawn on day 8 post infection in order to assess hematological and biochemical variables. Among the main results are: (1) animals from group C showed reduced erythrogram (with tendency to anemia); however the same results were not observed for group D; this might be a direct effect of free iron on trypanosomes which helped to reduce the parasitemia and the damage to erythrocytes caused by the infection; (2) iron supplementation was able to reduce NOx levels by inhibiting iNOS, and thus, providing an antioxidant action and, indirectly, reducing the ALT levels in groups Band D; (3) increase FRAP levels in group D; (4) reduce ADA activity in serum and erythrocytes in group C; however, this supplementation (5) increased the protein oxidation in groups B and D, as well as group C (positive control). Therefore, iron showed antioxidant and oxidant effects on animals that received supplementation; and it maintained the activity of E-ADA stable in infected/supplemented animals.

This study aimed to evaluate the Trypanosoma evansi susceptibility to tea tree oil (TTO - Melaleuca alternifolia) and tea tree oil nanocapsules (TTO nanocapsules) in vitro and in vivo tests. In vitro, we observed a mortality curve of trypomastigotes proportional to dose, i.e., the TTO and TTO nanocapsules have trypanocidal effect. Treatment with TTO in vivo was assessed in experiments (I and II). For Experiment I, T. evansi infected mice were treated with TTO and/or combinations of essential oil with chemotherapy (diminazene aceturate - D.A.). Treatment with TTO at a dose of 1mLkg(-1) was able to extend animal longevity, but had no curative efficacy. However, when TTO was combined with D.A. a disease curative efficacy of 100% for disease was observed, a much better result than the D.A. treatment (33.3%). In Experiment II, T. evansi infected mice were treated with TTO nanocapsules with doses of 0.3, 0.6 and 0.9mLkg(-1). Animals treated with 0.9mLkg(-1) showed higher longevity however without curative effect. Active compounds present in natural products, such as M. alternifolia, may potentiate the treatment of trypanosomosis when associated with other trypanocidal drugs.

The aim of this study was to investigate the susceptibility in vitro of Trypanosoma evansi to the essential oils of andiroba (Carapa guaianensis) and aroeira (Schinus molle), in their conventional and nanostructured forms. For that, pure oils at concentrations of 0.5%, 1.0% and 2.0% were used. A negative control (untreated) and a positive control (diminazene aceturate 0.5%) were used as comparative parameters. Later, the same tests were performed, using nanoemulsions oils at concentrations of 0.5% and 1.0%. The tests were carried out in triplicates and the numbers of parasites were quantified on 1, 3 and 6 h from onset of the study. A dose-dependent reduction in the number of parasites to the forms of two oils tested was observed after 1 h. The concentration of parasites was significantly reduced at low concentrations after 3 h, as well as at 6 h no alive parasites were observed for the essential oils tested. Ours findings indicate, for the first time, that oils of andiroba and aroeira (in their conventional and nanoemulsion forms) have high activity against T. evansi in vitro, leading to the suggestion that these oils may be applied as an alternative treatment for this disease.

The aim of this study was to evaluate biochemical parameters of iron metabolism in rats experimentally infected with Trypanosoma evansi. To this end, 20 rats (Wistar) were intraperitoneally inoculated with blood containing trypomastigotes 10(6) (Group T) and 12 animals were used as negative control (Group C) and received saline (0.2 mL) through same route. Blood samples were collected by cardiac puncture on day 5 (C5, T5) and 30 (C30, T30) post-inoculation (pi) to perform complete blood count and determination of serum iron, transferrin, ferritin, total and latent iron fixation capacity, transferrin saturation and prohepcidin concentration. Also, bone marrow samples were collected, to perform Pearls staining reaction. Levels of iron, total and latent iron binding capacity and prohepcidin concentration were lower (P

The aim of this study was to evaluate the nitric oxide (NO()) level, protein oxidation and antioxidant enzymes in rats infected with Trypanosoma evansi and establish the association of NO() levels with the degree of parasitemia. Thirty-six male rats (Wistar) were divided into two groups with 18 animals each. Group A was not infected while Group B was intraperitoneally infected, receiving 7.5×10(6) trypomastigotes per animal. Each group was divided into three subgroups with 6 rats each and blood was collected during different periods post-infection (PI), as follows: day 5 (A(5) and B(5)), day 15 (A(15) and B(15)) and day 30 PI (A(30) and B(30)). Blood samples were collected by cardiac puncture to estimate the levels of nitrites/nitrates (NO(x)) and advanced oxidation protein products (AOPP) in serum, and superoxide dismutase (SOD) and catalase (CAT) activities in blood. On days 15 and 30 PI NO(x) and AOPP levels were increased in serum of rats infected. Rodents infected with T. evansi showed a significant increase in SOD (days 5 and 15 PI) and CAT (day 30 PI) activities. Based on the physiological role of NO(), we can conclude that its increased concentration is related to an inflammatory response against the parasite, once a redox imbalance was observed during infection.

Rangeliosis is a disease which affects dogs in Brazil, caused by a piroplasm known as Rangelia vitalii. This disease causes a lot of clinico-pathological features, including the coagulation disorders associated with bleeding. The cause of these changes has not yet been determined. Considering the association of purinergic system and hemostasis this study aimed to evaluate the activity of enzymes that hydrolyze ATP, ADP and AMP; and deamination of adenosine in platelets from dogs experimentally infected with R. vitalii. For this study, 12 healthy young dogs (females) were used, separated in two groups. Group A (n=5) were uninfected controls, and group B were experimentally infected with R. vitalii (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite after five days post-inoculation (PI). Blood samples were collected to quantitate and separate platelets (Day 0, 12 and 21 PI) and to measure the enzymatic activities (Day 12 and 21 PI). The activity of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) was measured in platelets. A reduction (P

This study aimed to evaluate the adenine nucleotides and nucleoside concentration in serum and cerebral cortex of rats infected with Trypanosma evansi. Each rat was intraperitoneally infected with 1 × 106 trypomastigotes suspended in cryopreserved blood (Group A; n = 18). Twelve animals were used as controls (Group B). The infected animals were monitored daily by blood smears. At days 4 and 20 post-infection (PI) it was collected serum and cerebral cortex to measure the levels of ATP, ADP, AMP and adenosine by high performance liquid chromatography (HPLC). In serum there was a significant (P < 0.05) increase in the ATP, AMP and adenosine concentrations at days 4 and 20 PI in infected rats when compared to not-infected. Furthermore, in the cerebral cortex it was observed a significant (P < 0.05) increase in the concentrations of ATP, AMP and decreased adenosine levels at day 4 PI. At day 20 PI it was only observed an increase in the AMP and adenosine concentrations in cerebral cortex of infected rats when compared to not-infected. It was not observed any difference in ADP concentration in serum and brain at days 4 and 20 PI. No change was observed histologically in the cerebral cortex of infected animals. The results allow us to conclude that infection with T. evansi in rats causes an increase in the concentrations of ATP, AMP and adenosine in serum and cerebral cortex the time periods evaluated. These alterations occurred as a result of T. evansi infection which involves neurotransmission, neuromodulation and immune response impairment confirm the importance of the purinergic system in this pathology.

Nitric oxide (NO) is involved in many physiological processes, such as blood pressure control, neurotransmission, inhibition of platelet and neutrophil adherence, and the ability to kill tumor cells and parasites. The indirect determination of NO can be made by detection of 3-nitrotyrosine (3-NT) residues. The aim of this study was to measure the concentration of 3-NT in the brain of rats experimentally infected with Trypanosoma evansi. Twenty-four were inoculated intraperitoneally with cryopreserved blood containing 1×106 trypomastigotes per animal. Twenty-four animals were used as negative controls and received 0.2mL of saline by the same route. The experimental groups (group C and T) were established according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with six non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with six animals infected with T. evansi in each group. The animals were anesthetized with isoflurane and subsequently euthanized at the days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI). The brain was removed and dissected into cerebellum, cerebral cortex, striatum and hippocampus. Concentration of 3-NT in the brain was determined by Slot blot technique. At the day 3 PI no changes were observed in the concentration of 3-NT among the groups. There was a significant reduction (p

Recently we conducted the molecular characterization of Rangelia vitalii , a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected ( n = 5), and Group B consisted of animals infected with R. vitalii ( n = 7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B ( n = 6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs.

The aim of this study was to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1) and interleukin 6 (IL-6) in the serum of rats experimentally infected with Trypanosoma evansi and to correlate these levels with hematological parameters. Initially, 48 rats (group T) were intraperitoneally inoculated with cryopreserved blood containing 1×10(6) trypomastigotes per animal. Twenty-eight animals (group C) were used as negative controls and received 0.2 mL of saline by the same route. The experimental groups were formed according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with seven non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with 10 animals each inoculated with T. evansi. The blood samples were collected by cardiac puncture at days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI) to perform the complete blood count and the determination of IFN-γ, TNF-α, IL-1 and IL-6 levels using an ELISA quantitative sandwich. Infected rats showed normocytic normochromic anemia during the experimental period. T. evansi infection in rats caused a serum increase (P

The existence of cholinergic receptors in the immune system cells is well documented. This study aimed to evaluate the acetylcholinesterase activity (AChE) in lymphocytes from rats infected with Trypanosoma evansi in acute and chronic phase disease. Twenty animals were infected with 10(6) trypomastigotes forms each and 10 were used as negative controls. The two groups of inoculated rats were formed according to the degree of parasitemia and the period post-infection (PI). Group A: rats with 4 days PI and between 24 and 45 parasites/field (1000×); group B: rats with 30 days PI and parasitemia with jagged peaks between 0 and 1 parasites/field; group C: not-infected animals. At 4 days PI (acute phase) and 30 days PI (chronic phase) the rats were anesthetized to collect blood for hemogram and separation of lymphocytes. After separation, the AChE activity was measured in lymphocytes. It was observed that the number of lymphocytes increased significantly in group A compared to group C. The activity of AChE in lymphocytes significantly increased in acute phase and decreased in chronic phase in the infected rats when compared to not-infected (P0.05). Statistical analysis showed a positive correlation between the number of lymphocytes and AChE activity in lymphocytes in 4 days PI (r(2): 0.59). Therefore, the infection by T. evansi influences AChE activity in lymphocytes of rats indicating changes in the responses of cholinergic system in acute phase, possibly due to immune functions performed by these enzymes.

Duddingtonia flagrans is a nematode-trapping fungus responsible for attacking larval stages of helminths in pasture, which has potential as a biological control method. The aim of this study was to test the magnesium sulphate centrifugal flotation technique for the quantification of D. flagrans chlamydospores in sheep faeces and to verify their morphological viability. In this experiment one sheep received an oral dose of 4.5 × 10 6 chlamydospores/day during 20 days. Fecal samples were collected between days 15 and 20 and analyzed by the centrifugal flotation technique with magnesium sulphate. Densities of 1.23, 1.27 and 1.31 g mL −1 recovered 1.45 × 10 5 , 3.87 × 10 5 and 1.65 × 10 5 chlamydospores from the faeces, respectively. Based upon the results it was concluded that this is an efficient technique for the chlamydospores quantification in ovine faeces. Moreover, it allowed more accurate visualization of chlamydospore morphology.

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.

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (106 spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p

This study aimed to evaluate the activities of the ectoenzymes NTPDase and 5′-nucleotidase in synaptosomes from cerebral cortex of rats experimentally infected with Trypanosoma evansi. The animals were divided in four groups (n=10) according to the time and degree of parasitemia (groups A, B, C and D). The animals from group A were euthanized on day 3 (low parasitemia), group B on day 5 (high parasitemia) and group C on day 15 (low parasitemia). Group D consisted of healthy rats (not-infected, n=15) and were divided in three periods (n=5) in order to compare with the infected groups. After euthanasia, cerebral cortex was removed for the preparation of synaptosomes and enzymatic assays. Group A showed no changes in enzymatic activities compared with control. The hydrolysis of ATP, ADP and AMP by the enzymes NTPDase and 5′-nucleotidase were increased (P