Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Viability of nerve grafts preserved in different storage medium: ultrastructural features

We investigated the ultrastructural organization of transplanted autologous grafts after storage in two different solutions. Male Wistar rats were divided into groups to obtain normal tibial nerves, freshly transplanted nerves, and nerves stored in Wisconsin/Belzer or Collins solution for 24 or 72 hours at 4 °C and transplanted (W1, W3, C1, C3). After storage or transplantation, the specimens were processed for ultrastructural analysis. All grafts showed alterations in collagen fiber organization in the endoneurial space compared to normal nerves. These fibers were more loosely organized among nerve fibers, a finding that was significantly more marked in group C3 compared to groups W1 and W3. Important alterations were also observed in the myelin sheath structure of grafts stored in the two media. These changes were characterized by separation of the lipid lamellae, clearly visible in larger diameter nerve fibers. These findings were more marked and frequent in the C1 and C3 groups compared to the W1 and W3 groups. Ultrastructural analysis showed better preservation of Schwann cells and other elements that support axonal regeneration for grafts stored in Wisconsin/Belzer solution. These results support ongoing studies for the formulation of storage solutions that permit the creation of nerve banks for heterologous transplantation.

Early phase morphological lesions and transcriptional responses of bovine ileum infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5-12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric ss-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

Actin cytoskeletal and functional studies of the proximal convoluted tubules after preservation.

BACKGROUND: Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. METHODS: In vitro microperfusion technique and immunohistochemistry analysis. RESULTS: 1. Absorptions were similar in 1- and 24-hour-preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. CONCLUSION: There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.

Acute chagasic myocardiopathy after orthotopic liver transplantation with donor and recipient serologically negative for Trypanosoma cruzi: a case report.

Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi. Chagas disease following solid-organ transplantation has occurred in Latin America. This report presents the occurrence of Chagas disease despite negative serological tests in both the donor and the recipient, as well as the effectiveness of treatment. A 21-year-old woman from the state of Sao Paulo (Brazil) underwent cadaveric donor liver transplantation in November 2005, due to cirrhosis of autoimmune etiology. Ten months after liver transplantation, she developed signs and symptoms of congestive heart failure (New York Heart Association functional class IV). The echocardiogram, which was normal preoperatively, showed dilated cardiac chambers, depressed left ventricular systolic function (ejection fraction = 35%) and moderate pulmonary hypertension. Clinical investigation discarded ischemic heart disease and autoimmune and other causes for heart failure. Immuno fluorescence (immunoglobulin M and immunoglobulin G) and hemagglutination tests for T cruzi were positive, and abundant T cruzi amastigotes were readily identified in myocardial biopsy specimens. Treatment with benznidazole for 2 months yielded an excellent clinical response. At the moment of submission, the patient remains in functional class I. This case highlighted that more appropriate screening for T cruzi infection is mandatory in potential donors and recipients of solid-organ transplants in regions where Chagas disease is prevalent. Moreover, it stressed that this diagnosis should always be considered in recipients who develop cardiac complications, since negative serological tests do not completely discard the possibility of disease transmission and since good results can be achieved with prompt trypanocidal therapy.

Serum vitamin A levels in patients with ocular lesions attributable to non-complicated malaria in the Brazilian Amazon region.

In order to evaluate the association between serum vitamin A levels and ocular lesions attributable to non-complicated malaria, 200 patients seen consecutively at the Malaria Outpatient Clinic of FUNASA, Manaus, Amazonas, Brazil were included in this study. Ophthalmologic examination consisted of indirect binocular ophthalmoscopy under medicamentous mydriasis, biomicroscopy with a portable slit lamp and measurement of central visual acuity. Vitamin A serum concentration was determined by HPLC, and deficiency was defined as serum values equal to or lower than 0.35 micromol/l. Serum vitamin A values between 0.36 and 0.70 micromol/l were considered as marginal levels. Hypovitaminosis A (

Ocorrência de babesiose canina no Município de Uberlândia, Minas Gerais / Incidence of canine babesiosis in Uberlândia, Minas Gerais

Babesia canis é um protozoário intraeritrocítico transmitido pelo carrapato Rhipicephalus sanguineus que acomete principalmente canídeos domésticos e silvestres, causando manifestações clínicas como anorexia, pirexia, hemoglobinúria, esplenomegalia, hemoglobinemia, anemia grave, podendo o animal recuperar-se lentamente ou morrer. O objetivo desta pesquisa foi determinar a ocorrência da doença em Uberlândia – MG e comparar os métodos de esfregaços sangüíneos de ponta de orelha e Reação de Imunofluorescência Indireta (RIFI) no diagnóstico desta enfermidade. Para realização deste trabalho, foram colhidos aleatoriamente soros de 143 cães atendidos no Hospital Veterinário da Universidade Federal de Uberlândia para exame de RIFI enquanto que 772 esfregaços sangüíneos de ponta de orelha foram feitos e corados pelo Giemsa para a pesquisa do hemoparasito. Após a padronização da RIFI, considerou-se positivo soros com diluição igual ou superior a 1:20 e como negativos soros com diluição inferior a 1:20. Dos soros testados, através da RIFI, 51,74% estavam positivos e 48,26% negativos. Dos esfregaços sangüíneos 2,72% eram positivos para babesiose, 87,83% negativos e 9,45% positivos para outros hemoparasitos. Através do teste Qui-quadrado observou-se diferença significativa, em nível de significância 5%, entre os métodos comparados.

Campilobacteriose genital bovina em rebanhos leiteiros com problemas reprodutivos da microrregião de Varginha - Minas Gerais / Bovine genital campylobacteriosis in dairy herds with reproductive problems of the microregion of Varginha, MG, Brazil

The frequency of bovine genital campylobacteriosis (BGC) in 157 cows from nine herds from the microregion of Varginha - Minas Gerais - Brazil was evaluated. Farmers use either artificial insemination and natural breeding after two insemination procedures or natural breeding. The diagnosis of BGC was performed by the direct fluorescent antibody test (DFAT) using vaginal mucus. All studied herds showed C. fetus infected animals and, of 157 animals, 40 (25.5 percent) were positive in DFAT: 7 (26.9 percent) from herds with natural breeding and 33 (25.1 percent) from herds with both artificial insemination and natural breeding after the second-to-third insemination. The high frequency of BGC found in this study shows that this disease is present among herds which have reproductive problems and the use of natural breeding after the second-to-third unsuccessful insemination could be a risk factor for the disease

[Survival and infectious processes in pacients with AIDS: analysis based on initial serum vitamin A levels]. / Sobrevida e processos infecciosos em pacientes com AIDS: análise de acordo com os níveis séricos iniciais de vitamina A.

Patients with Aids (n = 39) were followed up for a maximum period of 36 weeks, after which the types and topographies of infectious complications presented and patient survival were analyzed and correlated with the vitamin A levels presented by the patients at the beginning of clinical follow-up. Twenty-one (53,8%) patients presented serum retinol levels below 1.6 micromol/L, 12 (57%) of whom had values lower than 1.05 micromol/L. There was no correlation between low serum vitamin A levels and the types or topographies of the infectious complications that occurred during the follow-up period. Although mean survival at the end of the 36 months follow-up period was similar for the two groups, patients with retinol deficiency presented a lower probability of survival during the first 24 months of follow-up compared to patients without hypovitaminosis A (8.44 x 1.42 months; p = 0.003).

[Toxoplasmosis of the central nervous system in a patient without immunosupression: case report]. / Toxoplasmose do sistema nervoso central em paciente sem evidência de imunossupressão: relato de caso.

The clinical manifestations of acquired toxoplasmosis in the immunocompetent patient rarely include localized neurological signs, which are frequent in the immunosuppressed patient (Aids). The objective of this paper is to report the case of a woman with Toxoplasma gondii brain abscess, without an identified cause of immunosuppression.

Seroprevalence of Toxoplasma gondii infection in goats by the indirect haemagglutination, immunofluorescence and immunoenzymatic tests in the region of Uberlândia, Brazil.

A comparative study of the indirect haemagglutination (IHA), immunofluorescence (IFAT) and immunoenzymatic (ELISA) tests was carried out to determine the prevalence of Toxoplasma gondii antibodies in goats. One hundred seventy-four serum samples were obtained from four goat herds from the region of Uberlândia, State of Minas Gerais. The distribution of the animals, according to their origin, was as follow: 71 from herd I; 39 from herd II; 37 from herd III; and 27 from herd IV. Serum samples were analyzed by IHA, IFAT and ELISA, considering the reactivity of the serum samples at dilution > or = 1:64 as cut off titer for the three tests. A global seroprevalence of 18.4% was observed, with significantly higher positivity rate in the herd II (66.7%) and older animals (> 36 months). A high and significant positive correlation was found between the titers obtained by the IHA versus IFAT, IHA versus ELISA, and ELISA versus IFAT. Therefore, it can be concluded that the three analyzed tests have shown to be highly concordant and appropriate for epidemiological surveys of Toxoplasma infection in goats. Although the seroprevalence of T. gondii infection in goats is relatively low in this region as compared to other regions of the country, adequate management might be useful and essential to control the infection in the goat herds.

[Serological markers of hepatitis B in users of services at an HIV Testing Center]. / Marcadores sorológicos da hepatite B em usuários de um Centro de Testagem para o HIV.

The objective of this investigation was to study the prevalence of serological markers of hepatitis B and possible risk factors for this disease in a sample of 404 people who attended a Testing and Couseling Center for HIV in the city of Ribeirão Preto, São Paulo State, Brazil. The overall prevalence of serologic hepatitis B markers was 14.6%, equal to that obtained for anti-HBc. HBsAg and anti-HBc IgM showed prevalences of 1%. After adjustment using logistic regression, hepatitis B markers showed association with the following variables: age, place of residence, use of injectable drugs and positivity to anti-HIV. The overall prevalence of human immunodeficiency virus infection was 6.9%. Hepatitis B markers were detected in 55.6% among intravenous drug users and in 42.9% among those who tested positive for HIV, confirming literature findings which indicates high levels of infection in these specific population groups.

Marcadores sorológicos da hepatite B em usuários de um Centro de Testagem para o HIV / Serological markers of hepatitis B in users of services at an HIV Testing Center

The objective of this investigation was to study the prevalence of serological markers of hepatitis B and possible risk factors for this disease in a sample of 404 people who attended a Testing and Couseling Center for HIV in the city of Ribeirão Preto, São Paulo State, Brazil. The overall prevalence of serologic hepatitis B markers was 14.6%, equal to that obtained for anti-HBc. HBsAg and anti-HBc IgM showed prevalences of 1%. After adjustment using logistic regression, hepatitis B markers showed association with the following variables: age, place of residence, use of injectable drugs and positivity to anti-HIV. The overall prevalence of human immunodeficiency virus infection was 6.9%. Hepatitis B markers were detected in 55.6% among intravenous drug users and in 42.9% among those who tested positive for HIV, confirming literature findings which indicates high levels of infection in these specific population groups.

Esta investigação objetivou estudar a prevalência de marcadores sorológicos de infecção pelo vírus da hepatite B e analisar possíveis fatores de risco em 404 usuários submetidos à sorologia anti-HIV no Centro de Testagem e Aconselhamento de Ribeirão Preto, SP, Brasil. A prevalência global dos marcadores para o vírus da hepatite B foi de 14,6%, idêntica à encontrada para o anti-HBc, com valores de 1% para o HBsAg e anti-HBc IgM. Após ajuste por regressão logística, os marcadores de infecção do vírus B mostraram associação com as variáveis: idade, local de residência, uso de drogas endovenosas e positividade para o HIV. A prevalência de infecção pelo vírus da imunodeficiência humana foi de 6,9%. Marcadores do vírus B foram detectados em 55,6% dos usuários de drogas endovenosas e em 42,9% dos positivos ao vírus da imunodeficiência humana, confirmando altos índices de infecção nestes grupos específicos.

Serum cytokines in patients with Brazilian pemphigus foliaceus (fogo selvagem).

Pemphigus foliaceus (PF) is characterized by acantholysis determined by IgG4 binding to desmoglein I, a 160-kDa desmosomal glycoprotein. To investigate the immunopathological aspects of Brazilian PF, we determined levels of serum cytokines in patients with PF. Twenty-five patients with PF and a control group consisting of 10 healthy individuals were studied. Serum IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma were measured in the two groups by ELISA. The median concentration of IL-2 was lower in PF patients compared to the control group (0.45 and 9.50 pg/ml, respectively), as also was the concentration of IL-4 (0.26 and 10.16 pg/ml, respectively). The same was observed for IL-5 (7.94 and 15.74 pg/ml, respectively) and for IFN-gamma (5.90 and 8.58 pg/ml, respectively). For IL-10 and IL-12, higher concentrations were observed in PF compared to the control group (IL-10: 24.76 and 20.92; IL-12: 2.92 and 1.17 pg/ml, respectively). Considering the Th1/Th2 paradigm, it seems that a Th2 profile, mainly represented by IL-10, predominates in PF.

Serum cytokines in patients with Brazilian pemphigus foliaceus (fogo selvagem)

Pemphigus foliaceus (PF) is characterized by acantholysis determined by IgG4 binding to desmoglein I, a 160-kDa desmosomal glycoprotein. To investigate the immunopathological aspects of Brazilian PF, we determined levels of serum cytokines in patients with PF. Twenty-five patients with PF and a control group consisting of 10 healthy individuals were studied. Serum IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma were measured in the two groups by ELISA. The median concentration of IL-2 was lower in PF patients compared to the control group (0.45 and 9.50 pg/ml, respectively), as also was the concentration of IL-4 (0.26 and 10.16 pg/ml, respectively). The same was observed for IL-5 (7.94 and 15.74 pg/ml, respectively) and for IFN-gamma (5.90 and 8.58 pg/ml, respectively). For IL-10 and IL-12, higher concentrations were observed in PF compared to the control group (IL-10: 24.76 and 20.92; IL-12: 2.92 and 1.17 pg/ml, respectively). Considering the Th1/Th2 paradigm, it seems that a Th2 profile, mainly represented by IL-10, predominates in PF

[Serological markers of hepatitis B in people submitted to blood testing in health care clinics]. / Marcadores sorológicos de hepatite B em indivíduos submetidos a exames de sangue em unidades de saúde.

OBJECTIVE: To study some of the epidemiological aspects of hepatitis B in a non-representative sample of patients seen in health care clinics. METHODS: The study population comprised 632 patients who were seen at health care clinics in the city of Ribeirão Preto, Brazil, for the purpose of blood testing, regardless the reason. After signing a written consent, an additional amount of blood was drawn from the same venous puncture site used to collect the original sample for the testing assigned to the patient at the health care clinic. A questionnaire was applied to each participant, looking for the presence of risk factors for hepatitis B. The blood samples were tested for HBV markers, using immunoenzimatic techniques. RESULTS: The prevalences of HBsAg and anti-HBcAg were 0.3% and 13.9%, respectively. By a logistic regression model, the following variables were significantly associated with the infection: age, time of residency in the city (higher risk among those living for a period less than one year), past history of hepatitis, incarceration and sexual behavior (higher risk among homosexual and bisexual males). CONCLUSIONS: The growing difficulties in obtaining blood samples from a representative group of patients, as done in classic surveys, make it necessary to look for alternative methodologies which can provide information concerning the presence of infectious agents in a community. Though the results cannot be generalized to the population as a whole, the methodology used conveyed some knowledge regarding the circulation of hepatitis B virus. In addition, it makes much easier to obtain agreement from the participants, since it does not add any invasive procedure. Despite the limitations, this methodology may be helpful in epidemiological surveillance of infectious agents known as producing asymptomatic infections in much of the population.

Production and characterization of a panel of monoclonal antibodies for the identification of antigens of Babesia bovis.

A panel of monoclonal antibodies was produced and characterized by an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and Western blotting with the aim of identifying antigens of Babesia bovis. After fusion, the resultant hybrids were selected by the IFAT, cloned, maintained in culture in vitro, and cryopreserved in liquid nitrogen. Ten clones producing monoclonal antibodies were found to react against the entire merozoites, three reacted on the surface of the merozoites, and one clone reacted against the polar region of the merozoites. All monoclonal antibodies reacted in ELISA, with the optical density varying from 0.368 to 0.502 (cut off = 0.022). The bands recognized by the monoclonal antibodies in Western blotting had molecular weights ranging from 162 to 19 kDa. Four clones recognized a single band of 73 kDa, and another four did not react in Western blotting.