Carlos Breno Paim

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

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.

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.

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.

Increased Cytokine and Nitric Oxide Levels in Serum of Dogs Experimentally Infected with Rangelia vitalii

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO x ) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NO x . Cytokines were assessed by ELISA quantitative sandwich technique, and NO x was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NO x were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (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 five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia.

Rangelia vitalii: changes in the enzymes ALT, CK and AST during the acute phase of experimental infection in dogs

Rangelia vitalii is a protozoon that causes diseases in dogs, and anemia is the most common laboratory finding. However, few studies on the biochemical changes in dogs infected with this protozoon exist. Thus, this study aimed to investigate the biochemical changes in dogs experimentally infected with R. vitalii, during the acute phase of the infection. For this study, 12 female dogs (aged 6-12 months and weighing between 4 and 7 kg) were used, divided in two groups. Group A was composed of healthy dogs (n = 5); and group B consisted of infected animals (n = 7). Blood samples were collected on days 0, 10, 20 and 30 after infection, using tubes without anticoagulant to obtain serum and analyze the biochemical parameters. An increase in alanine aminotransferase (ALT) on day 20 (P < 0.05) was observed. Also, increased creatine kinase (CK) and aspartate aminotransferase (AST) levels were observed throughout the experimental period (P < 0.05). No changes in the serum gamma-glutamyltransferase, urea and creatinine levels were observed. Thus, is possible to conclude that experimental infection with R. vitalii in dogs causes changes to the biochemical profile, with increased ALT, AST and CK enzyme levels.

Relationship between oxidative stress and clinical–pathological aspects in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate lipid peroxidation, protein oxidation and activity of enzymes that are indicators of oxidative stress in Rangelia vitalii infection in dogs. Animals were divided into two groups: negative control (n=5) and infected with R. vitalii (n=7). After inoculation, the parasitemia was estimated daily by microscopic examination of smears. Lipid peroxidation (TBARS) and advanced oxidation protein products (AOPP); and delta-aminolevulinate dehydratase (δ-ALA-D), superoxide dismutase (SOD) and catalase (CAT) activities in blood were evaluated. The samples were collected at days 10 and 20 post-inoculation (PI). TBARS and AOPP levels were higher in the infected group in both analyzed periods (P<0.01). The δ-ALA-D activity was reduced in blood of dogs infected with R. vitalii on days 10 and 20 PI. SOD activity was significantly increased (P<0.01) in the blood of dogs infected with R. vitalii at days 10 and 20 PI, while CAT activity was significantly increased (P<0.01) only at day 20 PI when compared to non-infected animals. A positive correlation was observed between the degree of parasitemia and TBARS and AOPP levels and activity of antioxidant enzymes. The δ-ALA-D activity was negatively correlated with the degree of parasitemia. Based on the increased levels of TBARS, AOPP, SOD and CAT activities, and inhibition δ-ALA-D activity, we concluded that dogs experimentally infected with R. vitalii develop a state of redox unbalance and that these changes might be involved in the pathophysiology of disease.

Serum proteinogram, acute phase proteins and immunoglobulins in dogs experimentally infected with Rangelia vitalii

The present study aimed to evaluate the serum proteinogram, acute phase proteins (APPs) and immunoglobulins (Igs) of dogs experimentally infected by Rangelia vitalii in the acute phases of the disease. Banked serum samples collected on days 0, 10 and 20 during a previously reported R. vitalii experimental infection were used to analyze the serum proteinogram, APPs (C-reactive protein - CRP and alpha-1-acid glycoprotein - AGP) and Igs (IgM, IgG, IgA and IgE) in the current study. Total protein and albumin level were significantly (P<0.05) decreased at day 10 PI and 20 PI in infected sera compared to the control sera. Alpha-1 globulin (day 10 PI) and gamma globulin (day 20 PI) were increased (P<0.01) in infected sera. Alpha-2 globulin (days 10 and 20 PI) and beta-2 globulin (day 10 PI) were decreased (P<0.05) in infected sera compared to control sera. Beta-1 globulin fraction did not differ statistically between sera. Serum CRP and AGP concentrations were significantly increased (P<0.05) at days 10 and 20 PI in infected sera. IgG was increased at days 10 (P<0.05) and 20 PI (P<0.01) in infected sera. Furthermore, it was also observed an increase (P<0.01) in the levels of IgM, IgA, and IgE in infected sera than control sera. We conclude that R. vitalii infection causes alterations in the proteinogram, and increases in the levels of APPs and Igs. Further studies are essentials to define the causes of these pathological changes in this disease.

Trypanosoma evansi: Concentration of 3-nitrotyrosine in the brain of infected rats

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 1×10(6) trypomastigotes per animal. Twenty-four animals were used as negative controls and received 0.2 mL 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<0.05) of 3-NT concentration in the striatum and cerebellum at the days 5 and 10 PI, respectively. At the day 20 PI a significant increase (p<0.05) of 3-NT was observed in the cerebellum, cerebral cortex and hippocampus from the infected animals. Therefore, T. evansi infection caused changes in the concentrations of 3-NT in the central nervous system (CNS), which may be related to clinical signs and infection management.

Influence of Rangelia vitalii (Apicomplexa: Piroplasmorida) on Copper, Iron, and Zinc Bloodstream Levels in Experimentally Infected Dogs

Abstract: The aim of this study was to evaluate the concentrations of copper, iron, and zinc in blood serum of dogs experimentally infected with Rangelia vitalii (n  =  7) compared with uninfected controls (n  =  5). Serum metal levels were determined in blood samples collected at days 0, 10, 15, and 20 post-infection (PI). Inductively coupled plasma optical emission spectrometry was used to measure the levels of copper, iron, and zinc. Significant differences (P < 0.05) were observed among groups PI. Increased levels of copper and decreased levels of iron and zinc were observed in the infected animals. The infection by R. vitalii may, therefore, alter the serum metal levels, resulting in metabolic disorders in dogs. These metals are directly involved in many enzymatic systems; accordingly, alterations in their blood concentrations may also influence the pathogenesis of disease.