Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Immunodiagnosis of swine cysticercosis by indirect ELISA employing a heterologous antigen from Taenia crassiceps metacestode

C. Larralde et al. (1990, Arch. Pathol. Lab. Med., 114:926-928) demonstrated that heterologous antigen from the laboratory-adapted murine Taenia crassiceps metacestode may substitute those from Taenia solium in the immunodiagnosis of human cysticercosis by the indirect enzyme-linked immunosorbent assay (IE). ?This antigen is easily obtained at a laboratory level and solves the problem of T. solium cysticerci collection from naturally or experimentally infected swine. In this study an IE employing a heterologous antigen from the T. crassiceps metacestode was evaluated for the immunodiagnosis of swine cysticercosis. Sera from 300 swine free of T. solium cysticerci by post-mortem examination were employed to determine two IE cut- off values: 1) Mean ELISA values + 2 standard deviations (2 sigma cut-off) and 2) - Mean ELISA values + 3 standard deviations (3 sigma cut-off). The specificity of IE was 97% with the 2 sigma cut-off and 100% with the 3 sigma cut-off. When applied to ten sera from swine infected by cysticerci of T. solium by post-mortem examination, the sensitivity of IE was 100% independent of the cut off.

Partial characterization of the polluting load of swine wastewater treated with an integrated biodigestion system

The stabilization of swine wastewaters from swine confined housing by the combination of a upflow anaerobic sludge blanket (UASB) reactor and waste stabilization ponds is a viable alternative to minimize the environmental impact caused by inadequate disposal of swine wastewaters. In the present study, the polluting load of pre-decanted swine wastewater treated with a series of two 0.705 m(3) UASB reactors and then in parallel in aerated and non-aerated stabilization tanks was investigated from January to July, 2000. Physicochemical and microbiological analyses were made adopting standard methods (Standard Methods for Examination of Water and Wastewater, 19th ed., American Public Health Association, Washington, DC, 1995). COD values decreased as the wastewater ran through the integrated biodigestion system dropping from about 3492 +/- 511-4094 mg l(-1) +/- 481 to 124 +/- 52-490 mg l(-1) +/- 230, while nitrate and nitrite levels increased in stabilization tanks, ranging respectively from 4 +/- 0 to 20 mg l(-1) +/- 3 and 3 +/- 1 to 11 mg l(-1) +/- 24. Although the removal of Escherichia coli was more than 97% +/- 6, the effluents of the treatment system still contained unacceptable levels of E. coli (1.6 x 10(3)-1.2 x 10(6) 100 ml(-1)) according to WHO guidelines for use of wastewater in agriculture and aquaculture. These results indicate the necessity of changes on operational characteristics of the treatment system such as an increase of the hydraulic retention time in UASB reactors or in stabilization tanks. (C) 2003 Published by Elsevier Ltd.

Partial genetic characterization of Stearoyl Coa-Desaturase's structural region in Bubalus bubalis

Conjugated Linoleic Acids (CLAs) comprise a family of positional and geometric isomers of linoleic acid. The main form of CLA, cis-9, trans-11-C18:2 show positive effects in cancer prevention and treatment. The major dietary sources of these fatty acids are derived from ruminant animals, in particular dairy products. In these animals, the endogenous synthesis mainly occurs in mammary gland by the action of enzyme Stearoyl CoA Desaturase (SCD). Different levels of expression and activity of SCD in mammary gland can explain partially the variation of CLA levels in fat milk. Considering a great fat concentration in bubaline milk and the benefit of a high and positive correlation between fat milk and CLA production, this study was carried on with the intention of sequencing and characterizing part of the gene that codifies SCD in buffaloes. Genomic DNA was extracted from blood samples of lactating bubaline which begins to the breed Murrah. After the (acho que nao precisa desse the) extractions, PCR (Polymerase Chain Reaction) reactions were made by using primers Z (sic) (sic) D1 and E1 (sic) (sic) F1. The fragments obtained in PCR were cloned into T vectors and transformed in competent cells DH10B line. After this, three samples of each fragment were sequenced from 5' and 3' extremities using a BigDye kit in an automatic sequencer. Sequences were edited in a consensus of each fragment and were submitted to BLAST-n / NCBI for similarity comparisions among other species. The sequence obtained with Z (sic) (sic) D1 primers shows 938 bp enclosing exons 1 and 2 and intron 1. The primers E1 (sic) (sic) F1 show 70 bp corresponding to exon 3 of bubaline SCD gene. Similarities were obtained between 85% and 97% among bubaline sequences and sequences of SCD gene described in human, mouse, rat, swine, bovine, caprine and ovine species. This study has permitted the identification and partial characterization of SCD codifing region in Bubalus bubalis specie.

Food intake and thermic effect of feeding in thyroid-deficient pigs

Short and long-term thyroidectomy and Methimazole treatment reduced food intake in young growing pigs. The thermic effect of feeding assessed by the increment in rectal temperature after the beginning of food ingestion was reduced in thyroidectomized animals, but no effect could be observed in Methimazole-treated pigs. Propranolol injection after short-term treatment decreased food intake in sham-operated and treated animals, but reduced the thermic effect of feeding only in the thyroidectomized and Methimazole-treated pigs. Long-term treatment inhibited the effect of propranolol in reducing food intake and the thermic effect of feeding. On the basis of these data, it was suggested that the interaction between thyroid hormones and catecholamines (noradrenaline) plays an important role in the regulation of food intake and in the thermic effect of feeding in thyroid-deficient pigs.

Observations on the development of the febrile response to pyrogen in newborn pigs

The febrile response of newborn pigs to exogenous pyrogen injection was investigated. Lipopolysacharides (LPS, E coli) were injected intravenously into the superior vena cava of 1-30-day-old piglets. All the experiments were carried out in littermates half of which were injected with pyrogen and half with pyrogen-free saline. Newborn pigs did not develop a febrile response from 1 to 4 days of age; however, when the animals were 5 days old exogenous pyrogen determined a typical monophasic febrile response. A second intravenous injection of pyrogen into newborn pigs (1 day old) 24 h after the first did not raise body temperature. It is suggested that newborn pigs behave like the newborn of other mammalian species regarding endotoxin-induced thermogenesis.

The fine structure of the swine sweat gland. II. The coiled ducts

The duct of the swine sweat gland crosses the dermis and epidermis in sequence. The cells of the dermic segment seem to be related with cellular secretion and absorption. In the epidermic segment of the duct the whole morphology of the cells resembles the cellular morphology of the epidermic cells.

Virulence factors in Escherichia coli strains isolated from pigs in the Ribeirao Preto region, State of Sao Paulo, Brazil.

Three-hundred faecal swabs were obtained from pigs with diarrhoea in farms located in different areas of the Ribeirao Preto region in the State of Sao Paulo. One-hundred Escherichia coli strains were isolated and tested for production of thermolabile (TL) and thermostable (STRa and STb) enterotoxins, and for the presence of colonization factors F4, F5 and F6. The strains were also tested for sensitivity to 14 antibiotics and chemotherapeutic agents. Twenty-four Escherichia coli strains produced enterotoxin STb, 5 produced LT and 3 produced STa. In the mannose-resistant haemagglutination reaction, one strain reacted positively with sheep, chicken, horse and human red blood cells and another reacted positively with guinea pig, sheep, chicken, horse and human red cells. However, both strains were negative for colonization factors F4, F5 and F6 when submitted to the slide agglutination test. All Escherichia coli strains were resistant to at least one antibiotic, the highest percentages being obtained for resistance to penicillin, tetracycline and cephalotin. In addition to the importance of the virulence factors normally encountered in enterotoxigenic Escherichia coli strains from pigs, the present results show the possible existence of new colonization factors other than F4, F5 and F6 participating in E. coli-induced pigs colibacillosis in the Ribeirao Preto region.

Toxicologic evaluation of acute and subacute oral administration of Cucurbita maxima seed extracts to rats and swine

The extract prepared from dried seeds of Cucurbita maxima was administered to rats and pigs. Following a single dose or 4 weeks of daily oral administration, the extract produced no changes in serum glucose, urea, creatinine, total protein, uric acid, GOT, GPT, LDH or blood counts. Urine analysis (urea, uric acid, creatinine, total protein, Na and K), as well as histopathological investigation, showed no abnormalities. These results taken as a whole indicate that the seeds of C. maxima as used in Brazilian folk medicine are not toxic for rats and swine.

Influence of different routes of flush perfusion on the distribution of lung preservation solutions in parenchyma and airways

Objective: The present study was performed to investigate the influence of different routes of perfusion on the distribution of the preservation solutions in the lung parenchyma and upper airways. Methods: Pigs were divided into four groups: control (n = 6), pulmonary artery (PA) (n = 6), simultaneous PA + bronchial artery (BA) (n = 8), and retrograde delivery (n = 6). After preparation and cannulation, cardioplegia solution and Euro- Collins solution (ECS) for lung preservation were given simultaneously. After removal of the heart, the double lung bloc was harvested. Following parameters were assessed: total and regional perfusion (dye-labeled microspheres), tissue water content, PA, aorta, left atrial and left ventricular pressures, cardiac output and lung temperature. Results: Our data show that flow of the ECS in lung parenchyma did not reach control values (9.4 ± 1.0 ml/min per g lung wet weight) regardless of the route of delivery (PA 6.3 ± 1.5, PA + BA 4.8 ± 0.9, retrograde 2.7 ± 0.9 ml/min per g lung wet weight). However, flow in the proximal and distal trachea were significantly increased by PA + BA delivery (0.970 ± 0.4, respectively, 0.380 ± 0.2 ml/min per g) in comparison with PA (0.023 ± 0.007, respectively, 0.024 ± 0.070 ml/min per g), retrograde (0.009 ± 0.003, respectively, 0.021 ± 0.006 ml/min per g) and control experiments (0.125 ± 0.0018, respectively, 0.105 ± 0.012 ml/g per min). Similarly the highest flow rates in the right main bronchus were achieved by PA + BA delivery (1.04 ± 0.4 ml/min per g) in comparison with 0.11 ± 0.03 in control, 0.033 ± 0.008 in PA, and 0.019 ± 0.005 ml/min per g in retrograde group. Flows in the left main bronchus were 0.09 ± 0.02 ml/min per g in control, 0.045 ± 0.012 ml/min per g in PA, and 0.027 ± 0.006 ml/min per g in retrograde group. The flow rates were significantly (P = 0.001) increased by PA + BA delivery of the storage solution (0.97 ± 0.3 ml/min per g). Conclusions: Our data show that the distribution of ECS for lung preservation is significantly improved in airway tissues (trachea and bronchi) if a simultaneous PA + BA delivery is used.

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type 11). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continues reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.

Acellular dermal tissue study: an ultrastructural evaluation of human and porcine derived tissues in a rat model

The purpose of this study was to evaluate the host response of a human and a porcine derived acellular dermal tissue (ADT) implanted in the subcutaneous tissue of a rat model. Two subcutaneous pockets were surgically created along the dorsal midline of 25 rats (5 rats/group). The human ADT was placed superiorly and the porcine ADT, inferiorly. The animals were sacrificed at 07, 15, 30, 60 and 180 postoperative days (PO) and the ADTs and surrounding soft tissues were assessed for ultrastructural evaluation by transmission electron microscopy. The ultrastructural findings were similar in both materials. Normal collagen and elastic fibers bundles were observed during all experimental moments, as well as macrophages presenting cytoplasmic enlargements digesting cellular portions after 15 PO. From 30 until 180 PO, vacuolar structures filled with an amorphous, electron-transparent substance, were present inside and outside the fibroblasts. Both human and porcine ADT showed similar pattern of ultrastructural response when implanted in the subcutaneous tissue of rats. The porcine ADT appears as a good alternative to be used as a biomaterial.