Andrey Pereira Lage

Synthesis and characterization of poly (vinyl alcohol) hydrogels and hybrids for rMPB70 protein adsorption

Polyvinyl alcohol (PVA), PVA crosslinked with glutaraldehyde hydrogels (PVA/GA), PVA with tetraethylorthosilicate (PVA/TEOS) and PVA/GA/TEOS hybrids with recombinant MPB70 protein (rMPB70) incorporated were chemically characterized by Fourier transform infrared spectroscopy (FTIR). FTIR spectra of PVA hydrogel samples showed the absorption regions of the specific chemical groups associated with poly(vinyl alcohol) (-OH, -CO, -CH2) and PVA/GA confirming the formation of crosslinked hydrogel (duplet -CH). It was observed C-H broad alkyl stretching band (n = 2850-3000 cm-1) and typical strong hydroxyl bands for free alcohol (nonbonded -OH stretching band at n = 3600-3650 cm-1), and hydrogen bonded band (n = 3200-3570 cm-1). The most important vibration bands related to silane alcoxides have been verified on FTIR spectra of PVA/TEOS and PVA/GA/TEOS hybrids (Si-O-Si, n = 1080 and n = 450 cm-1; Si-OH, n = 950 cm-1). FTIR spectra of f PVA hydrogel with rMPB70 incorporated have indicated the specific groups usually found in protein structures, such as amides I, II and III, at 1680-1620 cm-1, 1580-1480 cm-1 and 1246 cm-1, respectively. These results have given strong evidence that recombinant protein rMPB70 was successfully adsorbed in the hydrogels and hybrids networks. These PVA based hydrogels and hybrids were further used in immunological assays (Enzyme-Linked Immunosorbent Assay - ELISA). Tests were performed to detect antibodies against rMPB70 protein in serum samples from bovines that were positive in the tuberculin test. Corresponding tests were carried out without PVA samples in microtiter plates as control. Similar results were found for commercially available microplates and PVA based hydrogels and hybrids developed in the present work regarding to immunoassay sensitivity and specificity response.

Brucellosis in Brazil.

This paper reviews the epidemiology of bovine, swine, ovine, caprine, and canine brucellosis in Brazil. The zoonotic aspects of Brucella infection in Brazil is also discussed. Emphasis is given to the new program for the control of brucellosis in cattle and buffaloes that is likely to provide important insights into the prospects and strategies for controlling brucellosis in developing countries.

Pathogenesis of bovine brucellosis.

Bovine brucellosis is one of the most important zoonotic diseases worldwide, and is of particular significance in developing countries. The disease, which results in serious economic losses due to late term abortion, stillborn and weakly calves, is caused by Gram negative coccobacilli bacteria of the genus Brucella. Lesions consist of necrotic placentitis and interstitial mastitis in pregnant cows, and fibrinous pleuritis with interstitial pneumonia in aborted fetuses and newborn calves. This article considers the pathogenesis of Brucella abortus and reviews the ability of the pathogen to invade phagocytic and non-phagocytic host cells, resist the acidified intraphagosomal environment, and inhibit phagosome-lysosome fusion. Significant aspects of innate and adaptive immunity against brucellosis are also discussed.

Pathological, Immunohistochemical and Bacteriological Study of Tissues and Milk of Cows and Fetuses Experimentally Infected with Brucella abortus

This report describes a pathological, immunohistochemical and bacteriological study of 42 cows and their progeny (aborted fetuses, weak premature calves, and healthy full-term calves) infected at 6-7 months of gestation by conjunctival inoculation with Brucella abortus. Samples were collected at necropsy within 48 h of abortion or parturition. The most significant lesions were necrotizing and suppurative placentitis and lymphohistiocytic mastitis in cows, and fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia in aborted fetuses. B. abortus was isolated more frequently from milk samples than from mammary tissues, and milk samples from cows with mastitis were often infected. Organisms were often demonstrated immunohistochemically and by culture in tissues showing moderate to severe histological changes.

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

Modulation of the Bovine Trophoblastic Innate Immune Response by Brucella abortus

ABSTRACT Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis.

NRAMP1 3′ Untranslated Region Polymorphisms Are Not Associated with Natural Resistance to Brucella abortus in Cattle

ABSTRACT The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3′ untranslated region (3′UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3′UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCAr; n = 19) and in 43.1% of those with a susceptible genotype (SSCAs; n = 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCAr genotype were 86 and 84% in serologically positive (n = 64) and negative (n = 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3′UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.

Soroprevalência de aglutininas anti-Leptospira spp. em ovinos nas Mesorregiões Sudeste e Sudoeste do Estado Rio Grande do Sul, Brasil

The presence of anti-Leptospira agglutinins in 1.360 samples of ovine sera was determined. Clinically healthy sheep with more than one year of age, raised in pasture in 136 farms of 18 counties, 10 located in the southeast Mesorregions and 8 in the southwest Mesorregions of the state of Rio Grande do Sul, Brazil, between the months of January and March of 1999. Were used sera tested by the Microscopic Agglutination Technique (MAT), from the 1.360 samples of serum tested, 466 (34.26%) were positive and the titers of anti-Leptospira spp. agglutinins varied from 100 to 3.200. The serovars founded were: hardjo (Norma), 210 (28.4%), sentot, 152 (16.8%); hardjoprajitno, 133 (14.5%); fortbragg, 73 (6.3%); wolffi, 39 (4.7%); pyrogenes, 25 (1.8%); australis, 21 (1.6%); pomona, 20 (1.6%); sejroe, 19 (2.2%); castellonis, 18 (1.8%); hebdomadis, 17 (1.3%); icterohaemorrhagiae, 16 (0.5%); grippotyphosa, 9 (0.7%); canicola , 8 (0.6%); tarassovi, 7 (0.6%), bratislava, 4 (0.29%), autumnalis, 3 (0.2%). The results demonstrate that Leptospira spp are disseminated in the majority of the farms that raise sheep in the southeast and southwest Mesorregions of the State of Rio Grande do Sul, Brazil.

Detecção de cepas patogênicas pela PCR multiplex e avaliação da sensibilidade a antimicrobianos de Escherichia coli isoladas de leitões diarréicos

The frequency of virulence determinants genes for fimbrial adhesions (K88, K99, 987P, F18 and F41) and toxins (LT, Stb, StaP and Stx2e) in E. coli strains isolated from diarrheic piglets using the multiplex polymerase chain reaction assay with specific primers for these genes was studied. The antimicrobial sensitivity pattern of pathogenic isolates for florfenicol, sodium ceftiofur, colistin, fosfomycin, neomycin, norfloxacin, sulfa + trimetoprim, doxycycline, tetracycline and lincomycin was also tested using the disk diffusion method. E. coli were isolated from 144 diarrheic piglets from farms in the state of Minas Gerais. Forty-two out of 144 studied samples (29.2%) were positive for at least one tested virulence factor. Out of these 42, 23 samples (54.8%) contained fimbria and toxin genes, seven (16.6%) samples had genes for toxins only and 12 (28.6%) samples just fimbria genes. Disk diffusion in vitro antimicrobial sensitivity test demonstrated the best results for florfenicol (89.5%) and sodium ceftiofur (84.2%) against virulent E. coli strains.

Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams (n=9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks (n=40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.