Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Neospora caninum seropositivity in cattle breeds in the South Fluminense Paraíba Valley, state of Rio de Janeiro

The infection by Neospora caninum of different cattle breeds in dairy properties in two municipalities of the South Fluminense Paraíba Valley, state of Rio de Janeiro, was evaluated. Considering a sampling universe of 2,491 cows, blood samples were collected from 563 dairy cows in 57 farms, which were randomically selected in proportion to the number of animals, using a random stratified sampling system. For each property the number of selected cows was proportional to the herd size. Abortion or other reproductive disorders were not considered as criteria for selecting the animals, and seropositivity was determined by indirect enzyme-linked immunosorbent assay (ELISA). A high association (p=0.006) between seropositivity and racial patterns was found. The analysis revealed that in comparison of pure black-and-white Holstein cows versus Zebu (p=0.0028), Holstein cows showed 2.65 times greater odds for seropositivity. In the same way, by comparing black-and-white Holstein versus Zebu + crossbreed Zebu/Holstein (p=0.01), it was noted that there is 2.23 times more chance for seropositivity in Holstein cattle. There were no significant differences concerning the comparison of Holstein cattle versus crossbreed Zebu/Holstein (p=0.08) or Zebu versus crossbreed Zebu/Holstein (p=0.11). This study supports the hypothesis that there is a close association between cattle breeds and the frequency of infection by N. caninum.

Risk factors associated with the frequency of antibodies against Babesia bovis and Babesia bigemina in cattle in southern Mozambique

The study aimed to evaluate the risk factors associated with the frequency of IgG antibodies against Babesia bovis and B. bigemina in cattle in southern Mozambique. Eight hundred and nine serum samples were collected from cattle in three provinces namely Maputo, Gaza and Inhambane, and tested by indirect enzyme-linked immunosorbent assay (i-ELISA) to assess the humoral immune response towards B. bovis and B. bigemina. The chi-square test at 5% significance was used to determine whether there was an association between gender, age and geographic origin of seropositive animals. The overall prevalence was 78.8% (548/695) for B. bovis and 76.0% (528/695) for B. bigemina. The origin of the animals showed a significant association (p<0.05) with seropositivity to both agents, while gender and age was not associated (p>0.05). Maputo province had the highest rate of positive animals, with 93.7% (118/126) for B. bovis and 97.6% (123/126) for B. bigemina. In Gaza province 77.3% (321/415) of the animals were positive for B. bovis and 67.5% (280/415) for B. bigemina, while in the province of Inhambane the levels of seropositivity were 70.8% (109/154) and 81.2% (125/154) for B. bovis and B. bigemina respectively. In the present study, the frequency of cattle positive for B. bovis and B. bigemina was shown to increase among older age groups, suggesting that infection and re-infection persisted even after the primary infection. Thus, this region is considered to be in a state of enzootic stability with regards to B. bovis and B. bigemina.

Occurrence of infection with Toxoplasma gondii and factors associated with transmission in broiler chickens and laying hens in different raising systems

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. The aim of the present study was to determine the occurrence and identify the risk factors associated with transmission of T. gondii to chickens raised in different systems (free-ranged and confined) to produce eggs or meat. The 810 animals were allocated in two experimental groups according to the production system purpose: 460 broiler chickens (Group 1) and 350 layer chickens (Group 2). In order to analyze the possible factors involved in T. gondii infection in the chickens, an epidemiological questionnaire was developed for all properties.The serological detection of anti-Toxoplasma gondii antibodies was performed by Indirect Immunofluorescence (IFAT) and by Enzime Linked Imunossorbent Assay (ELISA). Since the agreement index (kappa) between these two serological techniques was considered high, 21.2% of the 810 animals were considered reactive. In Group 1, 12.2% (56/460) were positive, while in the Group 2 the positivity rate was 33.1% (116/350). The production system may be influencing the seropositivity of the animals in both groups. However, only in Group 2 it was possible to notice a statistically significant relationship between the breeding system and the frequency of positive sera. This result indicates that, at least for laying hens, the production system is directly involved in T. gondii infection. The contact with cats in Group 1 did not influence the distribution of seroreactive animals, but in Group 2 a significant relationship was observed. The occurrence of anti-T. gondii antibodies was high in both groups (broiler and posture chickens). Free-ranged chickens raised for egg production proved to be the most exposed group to the T. gondii infection. This can be related to the fact that these animals stay for longer periods in the farms, in direct contact with possibly contaminated soil by the presence of domestic cats.

Caracterização clínica e laboratorial de cães naturalmente infectados com Trypanosoma cruzi no semiárido nordestino

Objetivou-se, com este estudo, evidenciar os sinais clínicos e laboratoriais desta enfermidade para auxiliar na caracterização da doença de forma natural na área semi-árida da região nordeste. Foram avaliados 10 cães positivos para Trypanosoma cruzi, identificados mediante análises sorológicas de reação de imunofluorescência indireta (RIFI) e enzyme linked immunosorbent assay (ELISA); análise molecular pela Reação em Cadeia Polimerase (PCR), microscopia direta e hemocultura. Os cães chagásicos foram submetidos à avaliação física, verificação da pressão arterial, exames eletrocardiográficos, radiográficos, hematológicos (eritrograma e leucograma) e bioquímicos (ureia, creatinina, ALT, AST, PT, albumina, globulina, CK, CK-MB e cTnI). O exame físico e os valores das pressões arteriais dos cães apresentaram dentro dos parâmetros de normalidade, enquanto que na eletrocardiografia observou-se FC normal com ritmo sinusal, com exceção de um cão, que apresentou taquicardia sinusal (168 bat/min). No ECG de oito cães houve aumento da duração de P (47±6,5ms) sugestivo de aumento atrial, não confirmado radiograficamente. Foi observado supradesnivelamento do segmento ST em um cão. Nos resultados hematológicos constatou-se trombocitopenia (187,4x10³ ±137,2x10³) e anemia (5,0x10(6) ±1,39x10(6)/uL). Os valores médios da hemoglobina (11±2,7g/dL) e do hematócrito (34±10,5%) estavam abaixo dos limites de normalidade. A série branca apresentou-se dentro dos limites de normalidade, com exceção da eosinofilia observada em três cães. Individualmente, registrou-se em dois cães, leucocitose, linfocitose e neutrofilia. Na avaliação bioquímica, registrou-se hiperproteinemia (7,2±0,9g/dL), hipoalbuminemia (2,2±0,4g/dL), hiperglobulinemia (5,1±1,0g/dL) e aumento da CK (196±171U/L). Não houve alteração nas enzimas ALT e AST. A isoenzima CK-MB e o cTnI alteraram somente em três cães. Os cães infectados naturalmente no semiárido nordestino apresentam características relacionáveis à forma crônica indeterminada, ou seja, cães assintomáticos. A identificação dos cães infectados naturalmente sem características patognomônicas da doença de Chagas ressalta a importância desta enfermidade no processo diagnóstico com as demais que manifestam perfis inespecíficos associados ou não às doenças cardiovasculares.

Detection of cytomegalovirus infections by PCR in renal transplant patients

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4%) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50%) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Hepatitis C virus seroprevalence and genotypes in patients with diffuse connective tissue diseases and spondyloarthropathies

Many extrahepatic manifestations, including rheumatic diseases, have been reported to be associated with hepatitis C virus (HCV) infection. In order to investigate the prevalence of HCV infection among patients with rheumatic diseases, in the present study we interviewed 367 patients and tested their blood samples for HCV antibodies (anti-HCV) by an enzyme-linked immunosorbent assay. Anti-HCV-reactive samples were retested for confirmation by a line immunoassay and also for HCV RNA detection by the polymerase chain reaction. HCV RNA-positive samples were genotyped by INNO-LIPA. An overall HCV infection prevalence of 1.9% (7/367) was found. Of the 7 HCV-infected patients, 4 had systemic lupus erythematosus and 3 rheumatoid arthritis, resulting in positivity rates of 2.3 and 3.4%, respectively. HCV RNA genotyping revealed the presence of subtypes 1a (57.1%), 1b (28.6%) and 3a (14.3%). The clinical course was favorable for all HCV-infected patients, except one, who died due to renal insufficiency related to lupus nephritis. These results demonstrate a low HCV infection prevalence among the population studied. In the few positive cases, we observed no adverse influence of this infection on the clinical evolution of the rheumatic disease.

Serum and cerebrospinal fluid concentrations of E-selectin in patients with aneurysmal subarachnoid hemorrhage

The goal of the present study was to determine concentrations of E-selectin in both cerebrospinal fluid (CSF) and serum of patients with aneurysmal subarachnoid hemorrhage (SAH) and to evaluate the correlation between the clinical parameters and E-selectin levels. Both CSF and serum samples obtained from 12 patients with aneurysmal SAH and 8 patients with hydrocephalus (control group) without any other known central nervous system disease were assayed for E-selectin by quantitative enzyme-linked immunosorbent assay and the results were compared between the two groups. Mean levels of soluble forms of E-selectin within the first 3 days and on the 5th and 7th days of SAH were 4.0 ± 7.9, 2.8 ± 5.2, and 3.1 ± 4.9 ng/ml in the patient's CSF, and 33.7 ± 9.2, 35.1 ± 7.0, and 35.2 ± 8.7 ng/ml in serum, respectively. In contrast, mean E-selectin levels were 0.1 ± 0.2 ng/ml in CSF and 8.7 ± 5.0 ng/ml in serum of control patients. The difference between groups was statistically significant regarding both CSF and serum E-selectin levels (P < 0.05). Thus, we have demonstrated a marked increase of E-selectin concentration in both CSF and serum of patients with aneurysmal SAH compared with control and suggest that blocking the interaction between E-selectin and vascular endothelium may have a beneficial effect on vasospasms.

Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and β2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0%) hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95%CI: 21.5-25.4 MPL), only three carriers (<3%) had IgG anticardiolipin antibodies (median, 23 GPL; 95%CI: 20.5-25.5 GPL). Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004). IgA anti-β2-glycoprotein-I antibodies were detected in 29 of 109 (27.0%) hepatitis C carriers (median, 41 SAU; 95%CI: 52.7-103.9 SAU). Twenty patients (18.0%) had IgM anti-β2-glycoprotein I antibodies (median, 27.6 SMU; 95%CI: 23.3-70.3 SMU), while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU). Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-β2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

Leptin receptor (Ob-R) mRNA expression and serum leptin concentration in patients with colorectal and metastatic colorectal cancer

The objective of the present study was to investigate the effect of leptin on the progression of colorectal carcinoma to metastatic disease by analyzing the serum leptin concentration and Ob-R gene expression in colon cancer tissues. Tissue samples were obtained from 31 patients who underwent surgical resection for colon (18 cases) and metastatic colon (13 cases) cancer. Serum leptin concentration was determined by an enzyme-linked immunosorbent assay (ELISA) and Ob-R mRNA expression by real-time polymerase chain reaction (RT-PCR) for both groups. ELISA data were analyzed by the Student t-test and RT-PCR data were analyzed by the Mann-Whitney U-test. RT-PCR results demonstrated that mRNA expression of Ob-R in human metastatic colorectal cancer was higher than in local colorectal cancer tissues. On the other hand, mean serum leptin concentration was significantly higher in local colorectal cancer patients compared to patients with metastatic colorectal cancer. The results of the present study suggest a role for leptin in the progression of colon cancer to metastatic disease without weight loss. In other words, significantly increased Ob-R mRNA expression and decreased serum leptin concentration in patients with metastatic colon cancer indicate that sensitization to leptin activity may be a major indicator of metastasis to the colon tissue and the determination of leptin concentration and leptin gene expression may be used to aid the diagnosis.

Effects of single-dose atorvastatin on interleukin-6, interferon gamma, and myocardial no-reflow in a rabbit model of acute myocardial infarction and reperfusion

The mechanisms of statins relieving the no-reflow phenomenon and the effects of single-dose statins on it are not well known. This study sought to investigate the effects of inflammation on the no-reflow phenomenon in a rabbit model of acute myocardial infarction and reperfusion (AMI/R) and to evaluate the effects of single-dose atorvastatin on inflammation and myocardial no-reflow. Twenty-four New Zealand white male rabbits (5-6 months old) were randomized to three groups of eight: a sham-operated group, an AMI/R group, and an atorvastatin-treated group (10 mg/kg). Animals in the latter two groups were subjected to 4 h of coronary occlusion followed by 2 h of reperfusion. Serum levels of interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay. The expression of interferon gamma (IFN-γ) in normal and infarcted (reflow and no-reflow) myocardial tissue was determined by immunohistochemical methods. The area of no-reflow and necrosis was evaluated pathologically. Levels of serum IL-6 were significantly lower in the atorvastatin group than in the AMI/R group (P<0.01). Expression of IFN-γ in infarcted reflow and no-reflow myocardial tissue was also significantly lower in the atorvastatin group than in the AMI/R group. The mean area of no-reflow [47.01% of ligation area (LA)] was significantly smaller in the atorvastatin group than in the AMI/R group (85.67% of LA; P<0.01). The necrosis area was also significantly smaller in the atorvastatin group (85.94% of LA) than in the AMI/R group (96.56% of LA; P<0.01). In a secondary analysis, rabbits in the atorvastatin and AMI/R groups were divided into two groups based on necrosis area (90% of LA): a small group (<90% of LA) and a large group (>90% of LA). There was no significant difference in the area of no-reflow between the small (61.40% of LA) and large groups (69.87% of LA; P>0.05). Single-dose atorvastatin protected against inflammation and myocardial no-reflow and reduced infarct size during AMI/R in rabbits. No-reflow was not dependent on the reduction of infarct size.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.