Real-time quantitative PCR measurement of ileal Lactobacillus salivarius populations from broiler chickens to determine the influence of farming practices

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.     

Tissue Distribution and Metabolism of Aflatoxin B1-14C in Broiler Chickens

The effects of administering low levels of aflatoxin B(1)-(14)C by crop intubation daily for 14 days to broiler chickens were determined. Studies on the distribution of (14)C in the blood, selected organs, tissues, and excreta were conducted. No toxic effects were observed in broiler chickens during the 14 days of the experiment. The broiler chickens excreted 90.64% of the (14)C administered. Of the (14)C retained, 11.04, 9.83, 4.30, 12.52, 31.66, and 30.63% were detected in the blood, liver, heart, gizzard, breast, and leg, respectively. Chemical assay of those samples demonstrating radioactivity revealed that 81.2% of the radioactivity in these substrates was not extractable by classical extraction procedures while approximately 10% was extractable. Treatment of aqueous extracts for conjugated steroids by treatments with beta-glucuronidase revealed that 31.5% of the (14)C detected in the aqueous extract was a liberated glucuronide conjugate of aflatoxin M(1)-(14)C.     

Seroprevalence of Toxoplasma gondii in domestic and wild animals from the Fernando de Noronha, Brazil

Fernando de Noronha is an archipelago of 21 islands and islets in the Atlantic Ocean, state of Pernambuco, Brazil, which has a varied biodiversity including alien species or sinantropic animals. The objective here was to determine the seroprevalence of Toxoplasma gondii in domestic and wild animals from Fernando de Noronha archipelago, Brazil. Between July 2007 and May 2010, blood samples were collected from 764 animals (533 domestic and 231 wild animals). Sera were tested by the indirect fluorescence antibody test (IFAT) or the modified agglutination test (MAT), or by both. Antibodies to T. gondii were found in 80 (80.0%) of 100 chickens ( Gallus domesticus ), 3 (3.0%) of 100 cattle ( Bos taurus ), 59 (60.8%) of 97 sheep ( Ovis aries ), 9 (81.8%) of 11 goats ( Capra hircus ), 7 (43.7%) of 16 horses ( Equus caballus ), 70 (59.3%) of 118 cats ( Felis catus ), 36 (39.6%) of 91 dogs ( Canis familiaris ), 13 (38.2%) of 34 black rats ( Rattus rattus ), and 157 (79.7%) of 197 cattle egrets ( Bubulcus ibis ). Results indicate endemic infection by this zoonotic parasite among the animal and avian fauna in this archipelago from Brazil.     

Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma.

Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16-1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC(50)) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release.     

Heritability and genetic correlations of fear-related behaviour in Red Junglefowl--possible implications for early domestication

Domesticated species differ from their wild ancestors in a number of traits, generally referred to as the domesticated phenotype. Reduced fear of humans is assumed to have been an early prerequisite for the successful domestication of virtually all species. We hypothesized that fear of humans is linked to other domestication related traits. For three generations, we selected Red Junglefowl (ancestors of domestic chickens) solely on the reaction in a standardized Fear of Human-test. In this, the birds were exposed for a gradually approaching human, and their behaviour was continuously scored. This generated three groups of animals, high (H), low (L) and intermediate (I) fearful birds. The birds in each generation were additionally tested in a battery of behaviour tests, measuring aspects of fearfulness, exploration, and sociality. The results demonstrate that the variation in fear response of Red Junglefowl towards humans has a significant genetic component and is genetically correlated to behavioural responses in other contexts, of which some are associated with fearfulness and others with exploration. Hence, selection of Red Junglefowl on low fear for humans can be expected to lead to a correlated change of other behavioural traits over generations. It is therefore likely that domestication may have caused an initial suite of behavioural modifications, even without selection on anything besides tameness.     

Real-Time Quantitative PCR Measurement of Ileal Lactobacillus salivarius Populations from Broiler Chickens To Determine the Influence of Farming Practices

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.     

Investigation on possible ecotoxicological risk of carbofuran insecticides

Carbofuran-containing insecticides are widely used agents in plant protection. Their use may pose considerable environmental risk for both the protected and non-protected predator and plantivorous birds. For defence of wild birds a model experiment was carried out on broiler chickens. In the study, eight animals were treated orally by gastric tube with a carbofuran-containing insecticide at a single dose of 2.5 mg/kg b.w. One animal served as untreated control specimen. Forage and drinking water were provided ad libitum. After the treatment, the possible clinical signs were observed carefully, blood samples were obtained from each bird and after exsanguinations liver, breast and leg muscle samples and stomach content were taken. The carbofuran concentration in blood, tissues and stomach content was determined by gas chromatographic method. Thirty minutes after poisoning, the average carbofuran concentration in breast muscle of chickens exceeded the maximum level of 0.1 mg/kg permitted in edible tissues, whereas ninety minutes after poisoning the concentration of one sample was still above the limit value. In the liver, leg-muscle and blood samples, the measured carbofuran concentration was lower than the permitted maximum value, except in the blood of two animals. The carbofuran concentration of the stomach content markedly exceeded the limit value. The sublethal concentration of the pesticides can reduce the capable of living of wild animals. Due to the sub toxic dose the poisoned birds can survive; however, the residue of insecticides can lead to secondary toxicosis of other animals.     

Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 microl ml(-1) or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 degrees C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8.0 microl ml(-1) (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 10 degrees C. Aeromonas salmonicida strain MT004 (As MT004) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of 0.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity.     

Genetic relationships between Japanese native and commercial breeds using 70 chicken autosomal SNP genotypes by the DigiTag2 assay

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.     

Measuring circulating antioxidants in wild birds

Antioxidants protect against free radical damage, which is associated with various age-related pathologies. Antioxidants are also an important buffer against the respiratory burst of the immune system. This protection presumably has costs and therefore might underlie important life-history trade-offs. Studying such trade-offs in a comparative context requires field-applicable methods for assessing antioxidant capacity in wild animals. Here, we present modifications to a simple spectrophotometric assay (the TEAC or TAS assay) that can be applied to miniscule amounts of blood plasma to determine circulating antioxidant capacity. Additionally, uric acid, the most abundant circulating antioxidant, should be measured independently. Uric acid in birds is derived from amino acid catabolism, perhaps incidentally to its antioxidant function. The assay was validated in experimental studies on chickens showing effects of diet on antioxidant capacity, and in field measurements on 92 species of birds, which demonstrate substantial species differences in constitutive antioxidant capacity. Furthermore, most wild birds demonstrate a dramatic change in antioxidant capacity due to stress. These results show that this technique detects variation appropriate for both interspecific and intraspecific studies, and that antioxidants and uric acid change in response to conditions of interest to field ecologists, such as diet and stress.     

The effects of xylanase supplementation on performance, characteristics of the gastrointestinal tract, blood parameters and gut microflora in broilers fed on wheat-based diets

The aim of this study was to investigate the effects of xylanase supplementation on performance, characteristics of the gastrointestinal tract, blood parameters and gut microflora in broilers fed on wheat-based diets. The experimental diets consisted of a wheat-based diet supplemented with 0 or 1 g/kg enzyme preparation (xylanase activity was 1218 U/g). The diets were fed between 7 and 49 days of age. Enzyme supplementation (ES) improved (P<0.05) growth performance and feed conversion efficiency. The addition of enzyme to a wheat-based diet reduced the relative weights of the duodenum, jejunum, pancreas (P<0.05) and colon (P<0.01) in 21-day-old broiler chickens. Enzyme preparation reduced digesta viscosity in the proventriculus and jejunum of 21-day-old broiler chickens (P<0.05) and in colon of 49-day-old broiler chickens (P<0.05). The pH of the digesta in the crop, duodenum and jejunum was increased (P<0.05) in 21-day-old broiler chickens and was reduced in the caecum of 49-day-old broiler chickens (P<0.05) with enzymes. There was no significant difference between the two experimental groups in counts of lactobacillus and coliform bacteria in the caecum. Enzyme supplementation increased the concentration of blood insulin-like growth factor I (IGF-I) (P<0.01) of 21-day-old broilers, triiodothyronine (T₃) and insulin (P<0.05) at 49 days. ES reduced the concentrations of blood thyroxine (T₄) (P<0.01) and uric acid (P<0.05) at 49 days, but had no effect on glucose concentration (P>0.05). In conclusion, ES can improve performance and digestive parameters and can change some blood parameters in broiler chickens fed a wheat-based diet.     

Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus)

The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli-challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (10(6) cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 degrees C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH/GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH/GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH/GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH/GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens.     

Real-time PCR assay for the detection of species of the genus Mannheimia

Infections caused by species of the genus Mannheimia cause diverse disease complexes in many wild and domestic animals worldwide. Fast and accurate detection of single species within the genus remains an unsolved problem till today. To resolve this diagnostic challenge, we developed a real-time PCR assay for the rapid and specific identification of five species of the genus Mannheimia (M. haemolytica, M. varigena, M. ruminalis, M. granulomatis and M. glucosida) from bacterial cultures and tissue samples. The assay was validated with reference strains, field isolates and bacteria spiked tissue samples. The sodA gene was used as target region for species-specific primer pairs. The real-time PCR assay demonstrated species specificity for all five examined Mannheimia spp. and a rapid test completion time of less than 5 h. This is a considerable advantage compared to the traditional phenotyping methods currently used to distinguish between the species of the genus. The assay was able to detect approximately 10(3) bacterial cells per gram lung tissue sample, as determined with spiked tissue samples. We assume that the assay could become useful for fast laboratory diagnostic assessment particularly of respiratory infections caused by Mannheimia in animals.     

Endothermic heart rate response in broiler and White Leghorn chicks (Gallus gallus domesticus) during the first two days of post-hatch life

The embryonic modal value of heart rate (MHR) differs between broiler and White Leghorn chickens, but the initial development of cholinergic chronotropic control of embryonic heart rate (HR) does not. Thus, we hypothesized that hatchling MHR should also differ between broiler and White Leghorn strains, while the development of a physiological regulation, such as the endothermic HR response, should not be different between hatchlings of the two strains. To test this, we measured the response of HR and cloaca temperature (Tb) to alteration of ambient temperature (Ta); i.e., 35 degrees C-25 degrees C-35 degrees C, in four groups of hatchlings on Days 0 and 1 post-hatch. Fertile eggs of both strains with similar mass were incubated simultaneously in the same incubator. Eggs of broiler chickens hatched approximately 7 h earlier than White Leghorn chicken eggs. Chick mass at hatching was identical in both strains, but diverged during 2 days after hatching. Tb measured at the initial Ta of 35 degrees C was identical in both strains. MHR at the same Ta was approximately 30 bpm lower in broiler chicks than in White Leghorn chicks, but the difference was reversed to that observed in the embryos. The endothermic HR response was advanced by approximately 1 day in broiler chicks compared with White Leghorn chicks. As a result, eggs of similar mass in both strains produced chicks with similar mass and Tb at hatching, but during 2 days of post-hatch life their masses diverged and regulation of the endothermic HR response developed earlier in broiler than in White Leghorn hatchlings. This physiological heterochrony between strains is most likely due to genetic selection for fast growth in broiler chickens.     

The AB blood group system in wild felids

The blood type of 131 non-domesticated felids belonging to 26 felid species was surveyed in this study. Based upon a tube hemagglutination assay established for domestic cats, 80% of felids had type-A, 18% type-B, and 2% type-AB blood. Felids in the Puma group and African and Asian golden cats had blood type B, whereas all other species were found to have blood type A. Two cheetahs and one bobcat had type-AB blood. Red cell glycolipids analysed by high performance thin layer chromatography revealed a similar ganglioside pattern in wild cats as reported in domestic cats. Independent of the AB blood group system, incompatible blood crossmatch reactions were detected between different felid groups. In conclusion, wild felids display the same AB-erythrocyte antigens as domestic cats, and the same blood typing procedures can be applied for wild and domestic felids.     

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

ABSTRACT: BACKGROUND: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. RESULTS: Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6M and 3.3M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher's exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. CONCLUSION: A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122-1, 122-2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.     

Toxigenic fungi isolated from cereal and legume products

Summary An investigation of the toxigenicity of moulds recovered from domestic cereal and legume crops is described. Two hundred and twenty-eight mould strains, representing 59 species, were tested by feeding Pekin ducklings on maize meal infected with pure cultures. Forty-six strains, representing 22 species, caused death within 14 days. These 22 species were subsequently fed to weaned white mice and rats. Seven of these species caused death within 14 days, 6 showed less severe toxic effects while the remaining 9 gave no signs of acute poisoning in the animals.     

Effects of Corticosterone and Dietary Energy on Immune Function of Broiler Chickens

An experiment was conducted to investigate the effects of dietary energy level on the performance and immune function of stressed broiler chickens (Gallus gallus domesticus). A total of 96 three-day-old male broiler chickens (Ross × Ross) were divided into two groups. One group received a high energy (HE) diet and the other group received a low energy (LE) diet for 7 days. At 5 days of age, the chickens from each group were further divided into two sub-groups and received one of the following two treatments for 3 days: (1) subcutaneous injection of corticosterone, twice per day (CORT group; 2 mg of CORT/kg BW in corn oil) and (2) subcutaneous injection of corn oil, twice per day (Control/Sham treatment group). At 10 days of age, samples of blood, duodenum, jejunum, and ileum were obtained. Compared with the other three groups, the LE group treated with CORT had the lowest average daily gain (ADG) and the poorest feed conversion ratio (FCR, P < 0.05). Furthermore, CORT treatment decreased the relative weight (RW) of the bursa independent of the dietary energy level, but it decreased the RW of the thymus only in the chickens fed the LE diet. By contrast, CORT administration decreased the RW of the spleen only in the chickens fed the HE diet (P < 0.05). The plasma total protein, albumin, tumor necrosis factor alpha, interleukin 2 and immunoglobulin G (IgG) levels were affected by the CORT treatment (P < 0.05); however, these factors were not significantly affected by the dietary energy level. Toll-like receptor-5 mRNA level was down-regulated by CORT injection in the duodenum and ileum (P < 0.05) and showed a trend of down-regulation in the jejunum (P=0.0846). The present study showed that CORT treatment induced immunosuppressive effects on the innate immune system of broiler chickens, which were ameliorated by consumption of higher dietary energy.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

The kinetics and the distribution of antigen and antibody were shown to be similar in four species of experimental animals and in two species of wild rodents immunized with the protein-polysaccharide capsular plague antigen. Serologically active antigen and antibody were detected in homologous conjugating serological tests. Soluble antigen persists at the injection site for as long as a week and adsorbed antigen for two weeks or more. Antigen persists in the blood of animals for 2–4 days. In regional popliteal lymph nodes, antigen was detected for the first days, followed by antibody in both lymph node and blood. Plasma cell response was more intensive in animals inoculated with adsorbed antigen. The gradual decrease of antigen at the injection site shows superimposed up-and-down changes, mostly parallel with the antibody in the popliteal lymph node and blood, as well as with plasma cell response in the regional lymph node. Serological cycles were related to the resistance of immunized white mice to plague infection. Cyclic kinetics of specific polysaccharide in the faeces of dysentery patients was found.