Resistance to β-lactam and tetracycline in Campylobacter spp.isolated from broiler slaughterhouses in southern Brazil

Abstract The study was carried out to screen and analyze the genetic characteristics of antimicrobial resistance in Campylobacter spp. from poultry sources. A total of 141 strains of Campylobacter isolated from samples of broilers of slaughterhouses in southern Brazil was identified by phenotypic and genotypic methods. Campylobacter isolates were evaluated for its antimicrobial susceptibility and the presence of resistance genes. The strains were investigated for antimicrobial susceptibility against two agents (ampicillin and tetracycline) by disk diffusion method. PCR assay was used to confirm the specie and the presence of ampicillin (blaOXA-61), tetracycline tet(O), and the energy-dependent multi-drug efflux pump (cmeB) genes. Campylobacter jejuni was the most ubiquitous; its presence was determined in 140 samples out of 141 (99.3%), whereas Campylobacter coli was found only in one of the contaminated samples (0.70%). The results obtained showed 65% and 35.5% of Campylobacter isolates resistant to β-lactams and tetracyclines, respectively. The cmeB gene responsible for multidrug resistance was detected in 26 isolates out 141 strains (18.5%). Moreover, 36 out of 141 Campylobacter strains (25.6%) were found to be resistant to at least two different antimicrobia resistance markers (β-lactams and tetracyclines).

Effects of Cinnamon extract on biochemical enzymes, TNF-α and NF-κB gene expression levels in liver of broiler chickens inoculated with Escherichia coli

Abstract: Infection with Escherichia coli (E. coli) is a common disease in poultry industry. The use of antibiotics to treat diseases is facing serious criticism and concerns. The medicinal plants may be effective alternatives because of their multiplex activities. The aim of this study was to investigate the effects of cinnamon extract on the levels of liver enzymes, tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) gene expressions in liver of broiler chickens infected with E. coli. Ninety Ross-308 broilers were divided into healthy or E. coli-infected groups, receiving normal or cinnamon extract (in concentrations of 100 or 200mg/kg of food) supplemented diets. E. coli suspension (108cfu) was injected subcutaneously after 12 days cinnamon administration. Seventy-two hours after E. coli injection, the blood samples were taken for biochemical analysis of liver enzymes in serum (spectrophotometrically), and liver tissue samples were obtained for detection of gene expression of inflammatory markers TNF-α and NF-κB, using real-time PCR. Infection with E. coli significantly increased the levels of TNF-α and NF-κB gene expressions as well as some liver enzymes including creatine-kinase (CK), lactate-dehydrogenase (LDH), alanine-transferase (ALT) and aspartate-transferase (AST) as compared with control group (P<0.05). Pre-administration of cinnamon extract in broilers diet (in both concentrations) significantly reduced the tissue levels of TNF-α and NF-κB gene expressions and enzymes CK and ALT in serum of broiler chickens inoculated with E. coli in comparison with E. coli group (P<0.05 and P<0.01). The levels of LDH and AST were significantly decreased only by 200mg/kg cinnamon extract in infected broilers. The level of alkaline-phosphatase (ALP) was not affected in any groups. Pre-administration of cinnamon extract in diets of broiler chickens inoculated with E. coli could significantly reduce the gene expression levels of pro-inflammatory mediators and liver enzymes activities, thereby protecting the liver against this pathologic condition.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Influence of the neural tube/notochord complex on MyoD expression and cellular proliferation in chicken embryos

Important advances have been made in understanding the genetic processes that control skeletal muscle formation. Studies conducted on quails detected a delay in the myogenic program of animals selected for high growth rates. These studies have led to the hypothesis that a delay in myogenesis would allow somitic cells to proliferate longer and consequently increase the number of embryonic myoblasts. To test this hypothesis, recently segmented somites and part of the unsegmented paraxial mesoderm were separated from the neural tube/notochord complex in HH12 chicken embryos. In situ hybridization and competitive RT-PCR revealed that MyoD transcripts, which are responsible for myoblast determination, were absent in somites separated from neural tube/notochord (1.06 and 0.06 10-3 attomol MyoD/1 attomol ß-actin for control and separated somites, respectively; P<0.01). However, reapproximation of these structures allowed MyoD to be expressed in somites. Cellular proliferation was analyzed by immunohistochemical detection of incorporated BrdU, a thymidine analogue. A smaller but not significant (P = 0.27) number of proliferating cells was observed in somites that had been separated from neural tube/notochord (27 and 18 for control and separated somites, respectively). These results confirm the influence of the axial structures on MyoD activation but do not support the hypothesis that in the absence of MyoD transcripts the cellular proliferation would be maintained for a longer period of time.

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Cardioprotective effect of ornitho-kinin in an anesthetized, open-chest chicken model of acute coronary occlusion

The generation of bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) in blood and kallidin (Lys-BK) in tissues by the action of the kallikrein-kinin system has received little attention in non-mammalian vertebrates. In mammals, kallidin can be generated by the coronary endothelium and myocytes in response to ischemia, mediating cardioprotective events. The plasma of birds lacks two key components of the kallikrein-kinin system: the low molecular weight kininogen and a prekallikrein activator analogous to mammalian factor XII, but treatment with bovine plasma kallikrein generates ornitho-kinin [Thr6,Leu8]-BK. The possible cardioprotective effect of ornitho-kinin infusion was investigated in an anesthetized, open-chest chicken model of acute coronary occlusion. A branch of the left main coronary artery was reversibly ligated to produce ischemia followed by reperfusion, after which the degree of myocardial necrosis (infarct size as a percent of area at risk) was assessed by tetrazolium staining. The iv injection of a low dose of ornitho-kinin (4 µg/kg) reduced mean arterial pressure from 88 ± 12 to 42 ± 7 mmHg and increased heart rate from 335 ± 38 to 402 ± 45 bpm (N = 5). The size of the infarct was reduced by pretreatment with ornitho-kinin (500 µg/kg infused over a period of 5 min) from 35 ± 3 to 10 ± 2% of the area at risk. These results suggest that the physiological role of the kallikrein-kinin system is preserved in this animal model in spite of the absence of two key components, i.e., low molecular weight kininogen and factor XII.

Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of α and β transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. β2 integrin (CD18) associates with four different α (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that β2 integrin is also expressed by other types of cells. Since the gene for β2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that β2 integrin and the αL, αM, and αX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of β2 integrin or against its α subunit partners, showed that β2 integrin, as well as the αL, αM, and αX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of β2 integrin in these various locations in the embryonic heart. These results indicate that the β2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.

Proximate composition and quantification of fatty acids in breaded chicken steak

The aim of this work was to analyze the fatty acid and proximate composition of five brands of breaded chicken steak (A, B, C, D, and E) by accurate chromatographic quantification and to compare the experimental results with food label nutrition facts. The protein values of all brands were in agreement with the Brazilian regulation values, except for that of sample E, which presented the highest lipid content. Thirteen fatty acids were identified in the studied brands and the major ones were oleic acid, linoleic acid, and palmitic acid. The polyunsaturated fatty acid/saturated fatty acid ratios were within the values considered appropriate for human health; however, the high n-6/n-3 ratios found can result in an unbalanced intake of these fat acids. Only samples D and E can be considered trans free according to the regulations. The comparison of the analyses' results and the food label nutrition facts showed little variation in protein values. Nevertheless, most brands underestimated their lipid and energy levels. Brand B and C labels declared "free of trans", but the obteined results showed levels excedding those especified by regulation to be considered trans free values.

Modeling the water uptake by chicken carcasses during cooling by immersion

In this study, water uptake by poultry carcasses during cooling by water immersion was modeled using artificial neural networks. Data from twenty-five independent variables and the final mass of the carcass were collected in an industrial plant to train and validate the model. Different network structures with one hidden layer were tested, and the Downhill Simplex method was used to optimize the synaptic weights. In order to accelerate the optimization calculus, Principal Component Analysis (PCA) was used to preprocess the input data. The obtained results were: i) PCA reduced the number of input variables from twenty-five to ten; ii) the neural network structure 4-6-1 was the one with the best result; iii) PCA gave the following order of importance: parameters of mass transfer, heat transfer, and initial characteristics of the carcass. The main contributions of this work were to provide an accurate model for predicting the final content of water in the carcasses and a better understanding of the variables involved.

Sensory and microbiological evaluation of uncured fresh chicken sausage with reduced fat content

The purpose of this study was to evaluate the acceptability and the microbiological safety of uncured fresh chicken sausages with reduced fat content, considering the scientific evidence that correlated cancer and cardiovascular diseases to this diet. Two formulations of uncured fresh chicken sausage were processed using different concentrations of cochineal carmine pigment, rosemary extracts and synthetic antioxidants, which are used to give color, appearance and pleasant flavor to the products. Then, instrumental color (L*, a*, b*, C* and h*), microbial contamination and sensory tests (ranking and acceptance) were used to evaluate the quality of the uncured chicken sausages. The instrumental color (chroma and hue) and the sensory properties of the A and B uncured sausages were similar to the commercial cured sausage (C). However, the sensory color and appearance of samples A and B were statistically higher than those of the commercial uncured sausages D, which are prepared without the addition of nitrite or pigments. The results showed that it is possible to produce safe and high-quality uncured fresh chicken sausage with reduced fat content, using natural pigments and antioxidants.

Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Changes in breaded chicken and oil degradation during discontinuous frying with cottonseed oil

Discontinuous frying of breaded chicken in cottonseed oil was evaluated. Three 400 g batches of foodstuff were fried daily in a 28 L fryer at 182 °C for 4.5 minutes for 7-8 days, and the experiment was repeated three times. The total polar compounds in the oil were determined by the conventional method. Changes in the oil were determined by the quick tests Testo 265, Viscofrit and Fri-check based on physical constants, and the results were compared with those of total polar compounds obtained by the conventional method. The free fatty acids, conjugated dienes, Lovibond color, oxidative stability, fatty acid composition, and polymeric compounds were also determined. During frying, the oil samples presented 6.0-39.2% total polar compounds, 0.0-12.9% polymerized triacylglycerols, 1.3-14.5% oxidized triacylglycerols, 2.8-11.0% diacylglycerols, and 1.6-2.6% fatty acids and unsaponifiable polar compounds. The breaded chicken samples lost moisture, absorbed oil up to approximately 6%, and there were small changes in the fatty acid composition and low formation of trans-isomers. The best method for monitoring and discarding the oil was that used for the determination of total polar compounds.

Evaluation of the BAX® system for the detection of Salmonella spp. in naturally contaminated chicken meat

The aim of this study was to verify the efficiency of the BAX® system for the detection of Salmonella spp. in raw chicken meat. The conventional culture method (IN 62, MAP) was used as a reference method. A total of 8,813 chicken carcass samples were analyzed. In the first part of the study, 1,200 samples were analyzed using the BAX® System and the conventional culture method. In the second part, 7,613 samples were analyzed by the BAX® system, and the conventional method was used only for samples that tested positive for Salmonella spp. by the BAX® system. The sensitivity, specificity, relative accuracy, positive predictive value, and negative predictive value obtained in the first part of this study were 100%, 92.3%, 96.4%, 53.3% and 100%, respectively. The BAX® system showed no false-negative results and reduced the time to obtain presumptive positive results. It is a suitable method for use in laboratories that perform a large number of food samples analyses daily. However, the conventional method is still required to confirm the presence of Salmonella spp. in samples that test positive using the BAX® system.

Evaluation of quality factors of bovine and chicken meat marinated with reduced sodium content

In many industrialized countries, including Brazil, sodium intake exceeds the nutritional recommendations. Excessive consumption is associated with hypertension and premature death by cardiovascular diseases. The industry's challenge is to produce products with reduced sodium that are similar to regular products in texture and flavor and consistent with consumers' dietary habits. The present study aimed at substituting 25 and 50% NaCl for KCl in marinated beef and chicken meat with the addition of aromatic herbs and spices. The following microbiological analyses were carried out: macronutrient, chemical composition, and sensorial analysis. The meats showed a reduction in NaCl contents without any changes in their physical and chemical characteristics, and the products´ quality and microbiological safety were maintained. Beef and chicken tenderness was maintained for both treatments. Furthermore, the use of 50% KCl did not cause any changes in the products' sensory quality, and the overall acceptance of both types of meat was maintained. Results showed that a reduction by 50% in the NaCl contents of marinated meat products with a combination of herbs and spices is possible. Future applications in other meat products and sausages are highly promising.