Patrícia Wolkmer

Lipid peroxidation associated with anemia in rats experimentally infected with Trypanosoma evansi

This study aimed to assess the plasma lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during the early acute phase of Trypanosoma evansi infection in rats. Fifty male Wistar rats were randomly distributed into seven groups: three trypanosome-infected groups (T(2), T(4) and T(6); n=10 animals per group) and four uninfected controls (C(0), C(2), C(4) and C(6); n=5 animals per group). Animals from trypanosome-infected groups were inoculated intraperitoneally with 10(6) trypanosomes. Blood samples were collected by cardiac puncture before infection (day 0; group C(0)) or on the 2nd (C(2) and T(2)), 4th (C(4) and T(4)) and 6th (C(6) and T(6)) day post-infection (dpi). Samples were analyzed for red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), plasma malondialdehyde (MDA) and in vitro peroxidation of erythrocytes. The mean values of the hematological indices gradually decreased in the infected rats compared with the control. MDA was significantly increased (P<0.001) on the 6th dpi in infected versus control animals and was negatively correlated with PCV (P<0.001; R(2)=0.372). The values for erythrocyte in vitro peroxidation were higher for groups T(4) and T(6) than for the control rats (P<0.01). A positive correlation between erythrocyte peroxidation and MDA (P<0.001; R(2)=0.414) was observed. The results of this study indicate that T. evansi infection in rats is associated with oxidative stress, indicated by lipid peroxidation and oxidative damage in erythrocyte membranes, as demonstrated by in vitro peroxidation. This may be one of the causes of anemia in acute trypanosomosis.

Resposta eritropoética de ratos em diferentes graus de parasitemia por Trypanosoma evansi

O Trypanosoma evansi e um protozoario hemoflagelado que causa, em varias especies, uma doenca caracterizada por altos niveis de parasitemia, com rapido desenvolvimento de anemia. Este trabalho teve como objetivo investigar a relacao entre o grau de parasitemia e a alteracao na eritropoese de ratos (Rattus norvegicus) da linhagem Wistar infectados experimentalmente com T. evansi. Foram utilizados 42 ratos, dos quais 36 foram inoculados pela via intraperitoneal com 0,2ml de sangue, contendo 2,5 x 104 parasitas. Seis ratos nao-inoculados foram utilizados como controles. Apos inoculacao, a parasitemia foi avaliada a cada 12h. Os grupos para analise foram estipulados de acordo com a media de tripanossomas em 10 campos homogeneos focados aleatoriamente, sendo: A, controle; B, animais que apresentaram um grau de parasitemia entre 1-10 tripanossomas/campo; C, ratos com 11-20 tripanossomas/campo; D, ratos com 21-30 tripanossomas/campo; E, ratos com 31-40 tripanossomas/campo; F, 41-50 tripanossomas/campo; e G, ratos com mais de 51 tripanossomas/campo. Quando os animais apresentaram o numero de protozoarios equivalente ao grupo, foram coletadas amostras de sangue para realizacao de hemograma e dosagem de ferro, e foi realizada citologia de medula ossea para avaliacao da relacao mieloide:eritroide. A analise estatistica mostrou reducao significativa das hemacias e do hematocrito a partir de 31 tripanossomas/campo (grupos E, F e G; P<0,005) e a reducao de hemoglobina ocorreu a partir de 41 tripanossomas/campo (grupos F e G; P<0,005). A relacao mieloide:eritroide foi reduzida de 0,7 para 0,6 a partir de 41 tripanossomas/campo (grupos F e G; P<0,005). Nao foram detectadas variacoes na concentracao de ferro. Os dados obtidos demonstraram que ratos com parasitemia acima de 31 tripanossomas por campo desenvolvem uma anemia aguda, com um aumento compensatorio na atividade hematopoetica.

Cytokines in rats experimentally infected with Trypanosoma evansi

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<0.01) of IFN-γ, TNF-α, IL-1 and IL-6 levels at days 3, 5, 10 and 20 PI compared to the controls. The multiple linear regressions showed a reduction of 24% in the hematocrit as a consequence of the increased IFN-γ, TNF-α and IL-1. Therefore, we conclude that the infection caused by T. evansi causes an increase in the pro-inflammatory cytokines. These results suggest a synergism among IL-1, TNF-α and IFN-γ contributing to the development of anemia. This increase is associated with the regulation of immune responses against the parasite.

Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats.

Changes in blood, plasma and brain cholinesterase activities in Trypanosoma evansi-infected cats were investigated. Seven animals were infected with 10(8) trypomastigote forms each and six were used as control. Animals were monitored for 56 days by examining daily blood smears. Blood samples were collected at days 28 and 56 post-inoculation to determine the activity of acetylcholinesterase (AChE) in blood and the activity of butyrylcholinesterase (BChE) in plasma. AChE was also evaluated in total brain. The activity of AChE in blood and brain, and the activity of BChE in plasma significantly reduced in the infected cats. Therefore, the infection by T. evansi influenced cholinesterases of felines indicating changes in the responses of the cholinergic system.

Trypanosoma evansi: hematologic changes in experimentally infected 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 (P<0.05). 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.

Diminazene aceturate in the control of Trypanosoma evansi infection in cats.

The aim of this study was to investigate the efficacy of diminazene aceturate in the control of the infection by Trypanosoma evansi in cats. Fourteen animals were infected with 10(8) trypomastigote forms each and six were used as negative control (group A). Seven of the infected cats were used as positive control (group B) and seven were treated with diminazene aceturate (3.5 mg kg(-1)) for 5 consecutive days (group C). Biochemical and hematological parameters were evaluated during the experiment. Blood with anticoagulant was collected at day 49 post-inoculation and preserved in ethanol for DNA extraction. Samples were analyzed using PCR T. evansi-specific to assess the effectiveness of treatment. The treatment with diminazene aceturate had an efficacy of 85.7%. Alanine aminotransferase, gamma-glutamyltransferase, urea, and creatinine values remained within the normal physiological range in the treated cats. Hemogram was normalized in all the cured animals. Therefore, the therapy used is effective in controlling T. evansi in cats.

Cão naturalmente infectado por Trypanosoma evansi em Santa Maria, RS, Brasil

This paper describes the hematological alterations and proteinogram of a dog naturally infected by Trypanosoma evansi. This dog was presented with normochromic-normocytic anemia, leucopenia with lymphopenia followed for neutrophenia and lymphocitosis; and trombocitopenia. Hyperproteinemia with an increase of beta and gamma globulin fractions and hypoalbuminemia. By being the first case reported of T. evansi infection in dogs in Santa Maria, RS, Brazil, the epidemiological significance of such findings will alert the veterinarians to the existence of a possible parasites reservoir in the region warning to the possibility of new laboratory findings.

Trypanosoma evansi: cholinesterase activity in acutely infected Wistar rats.

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 25.86mU/lmolHb) compared with the control (37.84mU/lmolHb). In addition BUChE activity in plasma was lower in the T3 (7.01micromol BTC hydrolyzed/h/mL) than the T5 and C groups (9.84 and 12.00micromol 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.

Trypanosoma evansi: levels of copper, iron and zinc in the bloodstream of infected cats.

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 (P<0.05). 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.

Tripanossomose em equinos na região sul do Brasil

Background : : : : Trypanosoma evansi ( T. evansi ) is a protozoan which causes trypanosomosis in livestock in many countries of Southeast Asia, Africa and South America. Patterns of disease vary from acute epidemics with high case-fatality rates to subclinical and/or chronic disease in endemic animal populations. It is a problem of great economic importance due to the death of sick animals and high cost of treatment. This article aims to review the outbreaks of the infection by T. evansi in horses that occurred in southern Brazil. Review : These outbreaks were discussed in terms of epidemiology, clinical signs, laboratory tests, pathological findings, diagnosis and treatment by addressing the differences between the cases occurred in the state of Rio Grande do Sul and in other Brazilian states. The outbreaks due to T. evansi in livestock animals are endemic in warm-climate areas. At the Rio Grande do Sul state, most of the equine trypanosomosis occurs in the summer. This can be easily explained by the high number of bloodsucking insects, which are responsible for the mechanical transmission of the flagellate among the animals. Clinical signs such as progressive weight loss, lethargy, incoordination, instability, atrophy and paralysis of the hind limbs, difficulty in standing and walking, subcutaneous edema and abortion are often reported in T. evansi- infected equines. Anemia is the clinical alteration most observed in these infections, although its pathogenicity still remains unclear. In the present study anemia was associated with lipid peroxidation and decrease in serum iron levels and in acetylcholinesterase activity. Necropsy alterations found in the outbreaks reported in the Rio Grande do Sul state are commonly described in infections by T. evansi , except by the neurological alterations as necrotizing encephalitis of the white matter associated with edema, demyelinization and perivascular lymphoplasmocytic infiltrate. The diagnosis of the equine trypanosomosis was based on morphology and biometry of the trypomastigote forms in peripheral blood smears stained with Quick Panoptic or Giemsa methods, immunohistochemic, xenodiagnostic, and PCR T. evansi- specific. A new therapeutic protocol using diminazene aceturate at a dose of 7 mg kg -1 was tested in one of the outbreaks. This approach cured all the infected animals. Conclusion : Although T. evansi was diagnosed for the first time in Rio Grande do Sul state in 2002, veterinary clinicians have reported clinical signs such as paralysis of the pelvic members, fever and weight loss since the 80’s and 90’s. Therefore, the lack of knowledge of the disease might have been responsible for the unpublished data. Another hypothesis is the suspect of babesiosis, since both illnesses have marked anemia and hyperthermia. Moreover, as the diminazene aceturate has trypanocidal and babesicidal action, animals may have been misdiagnosed with babesiosis and may have recovered from the T. evansi infection. Only few researches on trypanosomosis are found in the southern region of Brazil. Prevalence studies with more sensible techniques are necessary in order to clarify the spread of the disease and the economic losses that it causes to farmers.