Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Subclinical muscle injuries in dogs infected with Leishmania (Leishmania) infantum chagasi

Although canine visceral leishmaniasis (CVL) has been extensively studied, muscular damage due to Leishmania (Leishmania) infantum chagasi infection remains to be fully established. The aim of this study was to describe the electromyographic and histological changes, as well as search for the presence of amastigote forms of Leishmania spp, CD3+ T-lymphocytes, macrophages and IgG in skeletal muscles of dogs with visceral leishmaniasis (VL). Four muscles (triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius) from a total of 17 naturally infected and six healthy dogs were used in this study. Electromyographic alterations such as fibrillation potentials, positive sharp waves and complex repetitive discharges were observed in, at least, three muscles from all infected dogs. Myocyte necrosis and degeneration were the most frequent muscular injury seen, followed by inflammatory reaction, fibrosis and variation in muscle fibers size. Immunohistochemistry in muscle samples revealed amastigote forms in 4/17 (23. 53%), IgG in 12/17 (70. 58%), CD3+ T-lymphocytes in 16/17 (94. 12%) and macrophages in 17/17 (100%) dogs. Statistically positive correlation was observed between: inflammatory infiltrate (p=0. 0305) and CD3+ immunoreaction (p=0. 0307) in relation to the number of amastigote forms; inflammatory infiltrate (p=0. 0101) and macrophage immunoreaction (p=0. 0127) in relation to the amount of CD3+; and inflammatory infiltrate (p=0. 0044) and degeneration/necrosis (p<0. 0001) in relation to the presence of macrophages. Our results suggest that different mechanisms contribute to the development of myocytotoxicity, including celular and humoral immune responses and direct muscular injury by the parasite. Nevertheless, the catabolic nature of the disease can probably interact with other factors, but cannot be incriminated as the only responsible for myositis.

Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs

The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n = 20) or very severe (i.e. with concurrent uremia; Leish IV, n = 20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n = 10) and to healthy dogs (Control, n = 30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease. © 2013 Elsevier Ltd. All rights reserved.

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp

Infected dogs are urban reservoirs of Leishmania chagasi, which is a causative agent of visceral leishmaniasis (VL). Dogs exhibit immune suppression during the course of this disease, and lymphocyte apoptosis is involved in this process. To investigate apoptosis and the expression levels of FAS-FAS-associated death domain protein (CD95 or APO-1), FASL-FAS ligand protein (CD178), and TRAIL-TNF-related apoptosis-inducing ligand (CD253) receptors in peripheral blood mononuclear cells and spleen leukocytes from 38 symptomatic dogs with moderate VL and 25 healthy dogs were evaluated by flow cytometry. The apoptosis rate of blood and splenic CD4+ and CD8+ cells was higher in infected dogs than in healthy dogs. The expression levels of FAS and FASL in blood and splenic CD4+ cells were lower in infected dogs than in healthy dogs. FAS expression in CD8+ cells was higher in infected dogs than in healthy dogs; in contrast, FASL expression was lower in infected dogs. The expression of the TRAIL receptor increased only in splenic CD8+ cells from infected dogs. The FAS and FAS-L blocking antibodies confirmed the importance of these receptors in apoptosis. Our results enhance the current understanding of the immune response in dogs infected with L. chagasi, facilitating the future development of therapeutic interventions to reduce lymphocyte depletion. © 2013 Elsevier B.V.

Prostatic acid phosphatase in serum and semen of dogs

The incidence of prostatic malignancy has increased the use of tissue markers to detect cancer. Tissue specific antigens or differentiation antigens are found on the surface of normal cells. Clinically, these antigens are important to diagnose alterations in the tissues and for immunotherapy. The objective of the present study was to evaluate the prostatic acid phosphatase concentration in blood and seminal plasma of intact and healthy dogs at different ages. The evaluation was carried out by spectrophotometer, using a commercial kit. The prostatic acid phosphatase (PAP) levels did not differ according to the age and did not correlate with age or prostatic dimensions verified by ultrasonography. The PAP concentration values varied greatly within each group. However, more studies are necessary to evaluate the role of prostatic acid phosphatase in the canine prostate and its importance as a diagnostic test for prostate disorders.

Assesment of the TEI index of myocardial performance in dogs with doxorubicin-induced cardiomiopathy

The development of a dose-dependent cardiomyopathy is the main limitation for the use of doxorubicin in chemotherapy protocols in both humans and animals. In this setting, the global myocardial function may be compromised resulting in signs of congestive heart failure. In this study, we investigated the ability of the Tei index of myocardial performance to identify myocardial dysfunction in healthy dogs receiving doxorubicin to a cumulative dose of 210 mg/m(2) over 147 days, comparing it with other standard echocardiographic indicators of systolic and diastolic function. Our results indicated that the Tei index, the isovolumic relaxation time, pre-ejection period and the pre-ejection period-to-left ventricular ejection time ratio were able to identify the cardiotoxic effects of doxorubicin on cardiac function when only 60 mg/m(2) had been administered, while the standard systolic and diastolic parameters, including left ventricular diameter at systole, ejection fraction, and fractional shortening needed at least 120 mg/mg(2) to deteriorate. We concluded that prolonged anthracycline therapy compromises both systolic and diastolic functions, which may be documented earlier by including the Tel index evaluation to the standard echocardiographic assessment of animals receiving doxorubicin.

Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel

Background: Infectious diarrhea can be caused by bacteria, viruses, or protozoan organisms, or a combination of these. The identification of co-infections in dogs is important to determine the prognosis and to plan strategies for their treatment and prophylaxis. Although many pathogens have been individually detected with real-time polymerase chain reaction (PCR), a comprehensive panel of agents that cause diarrhea in privately owned dogs has not yet been established. The objective of this study was to use a real-time PCR diarrhea panel to survey the frequencies of pathogens and co-infections in owned dogs attended in a veterinary hospital with and without diarrhea, as well the frequency in different countries. Feces samples were tested for canine distemper virus, canine coronavirus, canine parvovirus type 2 (CPV-2), Clostridium perfringens alpha toxin (CPA), Cryptosporidium spp., Giardia spp., and Salmonella spp. using molecular techniques.Results: In total, 104 diarrheic and 43 control dogs that were presented consecutively at a major private veterinary hospital were included in the study. Overall, 71/104 (68.3%) dogs with diarrhea were positive for at least one pathogen: a single infection in 39/71 dogs (54.9%) and co-infections in 32/71 dogs (45.1%), including 21/32 dogs (65.6%) with dual, 5/32 (15.6%) with triple, and 6/32 (18.8%) with quadruple infections. In the control group, 13/43 (30.2%) dogs were positive, all with single infections only. The most prevalent pathogens in the diarrheic dogs were CPA (40/104 dogs, 38.5%), CPV-2 (36/104 dogs, 34.6%), and Giardia spp. (14/104 dogs, 13.5%). CPV-2 was the most prevalent pathogen in the dual co-infections, associated with CPA, Cryptosporidium spp., or Giardia spp. No statistical difference (P = 0.8374) was observed in the duration of diarrhea or the number of deaths (P = 0.5722) in the presence or absence of single or co-infections.Conclusions: Diarrheic dogs showed a higher prevalence of pathogen infections than the controls. Whereas the healthy dogs had only single infections, about half the diarrheic dogs had co-infections. Therefore, multiple pathogens should be investigated in dogs presenting with diarrhea. The effects of multiple pathogens on the disease outcomes remain unclear because the rate of death and the duration of diarrhea did not seem to be affected by these factors.

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CD4+FOXP3+cells produce IL-10 in the spleens of dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serum DHEA-S increases in dogs naturally infected with Ehrlichia canis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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. (C) 2011 Elsevier B.V. All rights reserved.

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.

Topographic and age-dependent distribution of subchondral bone density in the elbow joints of clinically normal dogs

OBJECTIVE: To investigate topographic and age-dependent adaptation of subchondral bone density in the elbow joints of healthy dogs by means of computed tomographic osteoabsorptiometry (CTOAM). Animals-42 elbow joints of 29 clinically normal dogs of various breeds and ages. PROCEDURES: Subchondral bone densities of the humeral, radial, and ulnar joint surfaces of the elbow relative to a water-hydroxyapatite phantom were assessed by means of CTOAM. Distribution patterns in juvenile, adult, and geriatric dogs (age, < 1 year, 1 to 8 years, and > 8 years, respectively) were determined and compared within and among groups. RESULTS: An area of increased subchondral bone density was detected in the humerus distomedially and cranially on the trochlea and in the olecranon fossa. The ulna had maximum bone densities on the anconeal and medial coronoid processes. Increased bone density was detected in the craniomedial region of the joint surface of the radius. A significant age-dependent increase in subchondral bone density was revealed in elbow joint surfaces of the radius, ulna, and humerus. Mean subchondral bone density of the radius was significantly less than that of the ulna in paired comparisons for all dogs combined and in adult and geriatric, but not juvenile, dog groups. CONCLUSIONS AND CLINICAL RELEVANCE: An age-dependent increase in subchondral bone density at the elbow joint was revealed. Maximal relative subchondral bone densities were detected consistently at the medial coronoid process and central aspect of the humeral trochlea, regions that are commonly affected in dogs with elbow dysplasia.

Ground reaction force profiles after partial and pancarpal arthrodesis in dogs

OBJECTIVES: To evaluate and compare long-term functional outcome after partial carpal arthrodesis and pancarpal arthrodesis in dogs using kinetic gait analysis. METHODS: Fourteen dogs with 19 partial carpal or pancarpal arthrodeses were retrospectively examined and underwent force-plate gait analysis. Mean times since surgery were 29.4 and 24.4 months for pancarpal and partial carpal arthrodesis respectively. Vertical and braking-propulsive ground reaction force profiles were compared between treatment groups, and to those of normal dogs (control group) using Kruskal-Wallis one-way analysis of variance. RESULTS: With the exception of time to vertical peak that occurred earlier in dogs with pancarpal than in dogs with partial carpal arthrodesis (p <0.01), there was no difference between the two treatment groups. Several parameters differed significantly between operated and healthy dogs (p <0.01): vertical impulses were significantly lower in both treatment groups, braking forces and impulses were also reduced after both techniques. Propulsive forces and impulses were only reduced in dogs with pancarpal arthrodesis. When comparing gait parameters of sound limbs of unilateral operated dogs to those of control dogs, braking forces and impulses (p <0.01; p <0.05) were significantly higher in the sound legs of unilateral operated dogs. CLINICAL SIGNIFICANCE: Long-term outcome after partial carpal and pancarpal arthrodesis is good and comparable to each other. Propulsive action may be altered more in dogs with pancarpal arthrodesis.