Dietary supplementation with ferric tyrosine improves zootechnical performance and reduces caecal Campylobacter spp. load in broilers.

1. The objective of this study was to evaluate the effect of ferric tyrosine on the reduction of Campylobacter spp. and zootechnical performance in broilers exposed to Campylobacter spp. using a natural challenge model to simulate commercial conditions. Additionally, the minimum inhibitory concentrations (MICs) of ferric tyrosine against common enteropathogens were evaluated. 2. At the start of the trial, 840 healthy male 1-d-old birds (Ross 308) were randomly allocated to 6 replicate pens of 35 birds each and fed diets containing different concentrations of ferric tyrosine (0, 0.02, 0.05 and 0.2 g/kg) in mash form for 42 d. 3. Broilers fed diets containing ferric tyrosine showed significantly higher body weight at d 42 and weight gain compared to the control group. However, birds fed ferric tyrosine ate significantly more than the control birds so significant improvements in feed conversion rate were not observed. 4. Microbiological analyses of caecal samples collected on d 42 of the study showed, per gram of sample, 2-3 log10 reduction in Campylobacter spp. and 1 log10 reduction in Escherichia coli in the groups fed diets containing ferric tyrosine compared to the control. 5. The MICs of ferric tyrosine was >400 mg/l for C. jejuni and >200 mg/l for E. coli and Salmonella enterica, indicating that ferric tyrosine did not exert antimicrobial activity. 6. The results showed that birds fed ferric tyrosine grew faster and consumed more feed compared to the control group, indicating potential benefits of faster time to reach slaughter weight with no significant reduction on feed efficiency. Moreover, ferric tyrosine significantly reduced caecal Campylobacter spp. and E. coli indicating potential as a non-antibiotic feed additive to lower the risk of infections transmitted through the food chain.

Prophylactic administration of a combined prebiotic and probiotic, or therapeutic administration of enrofloxacin, to reduce the incidence of bacterial chondronecrosis with osteomyelitis in broilers.

Bacteria entering the bloodstream via translocation from the gastrointestinal tract spread hematogenously and can trigger bacterial chondronecrosis with osteomyelitis (BCO) by infecting osteochondrotic microfractures in the epiphyseal-physeal cartilage of the proximal femora and tibiae. In experiment 1, broilers were fed control feed or the same feed containing BacPack 2X, which includes the prebiotic IMW50 (a mannan oligosaccharide beta-glucan yeast cell wall product) plus the probiotic Calsporin (Bacillus subtilis C-3102). Broilers reared on wire flooring consistently developed higher incidences of BCO than hatchmates reared on wood shavings litter (≥24 vs. ≤4%, respectively; P=0.001). Adding BacPack 2X to the feed on d 1 through 56 delayed the age of onset and reduced the cumulative incidence of BCO on wire flooring when compared with broilers fed the control feed (24.0 vs. 40.7%, respectively; P=0.003). In experiment 2, broilers reared on wire flooring received tap water on d 1 through 62 (control group) or therapeutic levels of the potent fluoroquinolone antimicrobial enrofloxacin in the water on d 35 through 54 (enrofloxacin group). During enrofloxacin administration, half as many birds developed BCO in the enrofloxacin group when compared with the control group (8.1 vs. 19.5%, respectively, on d 35 through 54; P=0.001), whereas both groups had similar BCO incidences subsequent to withdrawing enrofloxacin on d 55 through 62 (14.8 vs. 18.2% for the enrofloxacin vs. control groups; P=0.386). Cumulative lameness incidences for d 1 through 62 were higher for the control group than for the enrofloxacin group (39.0 vs. 25.8%, respectively; P=0.003). These results demonstrate that wire flooring imposes a rigorous challenge that leads to high incidences of BCO that can be difficult to suppress, even with therapeutic doses of enrofloxacin. Prophylactically adding BacPack 2X to the feed reduced the incidence of BCO lameness by a proportion similar to that achieved with enrofloxacin, indicating that probiotics potentially can provide effective alternatives to antibiotics for reducing BCO lameness attributable to bacterial translocation and hematogenous distribution.

Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron.

Originally reported in 1983, channel catfish anemia (CCA), also 'white lip' or 'no blood,' is a major idiopathic disease affecting commercial production in the Mississippi Delta region of the USA. Affected individuals are characterized by lethargy, anorexia, extreme pallor, and packed cell volumes often below 5%, but a definitive cause for CCA remains elusive. Records from the National Warmwater Aquaculture Center (NWAC) reveal that, on average, CCA accounted for 4.7% of case submissions from 1994 to 2012. Known infectious agents, parasites, and perturbations in commonly measured water quality variables have been largely excluded, and research has focused on potential feed-related etiologies, particularly folic acid deficiency. No natural or anthropogenic contaminants have been found in feeds, and no associations have been made to any particular feed brand or formulation, or to the age or condition of the feed itself. Contrary to reports indicating a short clinical course, NWAC records indicate an insidious condition where certain ponds have contained fish diagnosed with CCA for up to 4 consecutive years and individual outbreaks have persisted for at least 5 mo. Investigation into the iron status of CCA-affected fish revealed values consistent with iron deficiency anemia, including low-packed cell volume (mean ± SE, 5.6 ± 1.0 vs. 24.8 ± 2.4%), serum iron (35.2 ± 3.5 vs. 104.4 ± 18.5 µg dl-1), liver iron (12.2 ± 2.6 vs. 23.3 ± 4.6 µg g-1), and percent transferrin saturation (14.5 ± 2.7 vs. 26.9 ± 3.1%) in anemic and healthy controls, respectively. Administration of parenteral iron produced complete recovery and returned iron indices to within the ranges of normal controls. Despite these findings, factors predisposing a state of hypoferremia remain unknown.

Comparative efficacy of flubendazole and a commercially available herbal wormer against natural infections of Ascaridia galli, Heterakis gallinarum and intestinal Capillaria spp. in chickens.

The efficacy of a commercially available flubendazole-based product and a commercially available herbal product were compared against three species of helminth parasites of chickens: Ascaridia galli, Heterakis gallinarum and Capillaria spp. A total of 48 naturally infected chickens were used in the study with 16 birds in each of three treatment groups (untreated control; flubendazole; and a herbal product). One bird from each treatment group was necropsied on Day 0 prior to first treatment to confirm the parasite species present in the birds. Treatments were administered as labelled and the 45 remaining birds were necropsied on Day 12 and worm counts performed. Average worm counts in the two treated groups were compared to the untreated controls to calculate efficacy. Flubendazole (Group A) achieved an overall efficacy of 99.4% for the three parasite species. The herbal product (Group B) achieved efficacies ranging from less than zero to 11.6% for the three parasites, with worm counts not significantly different to the untreated controls. At present, commercially available herbal products claiming anthelmintic properties do not require licencing as veterinary medicinal products (Directive 2004/28/EC: see Article 17 and 33-38) and thus are not required to meet specific efficacy thresholds. Products which do not appear to deliver acceptable anthelmintic efficacy, are obviously a concern from many aspects but specifically from an animal welfare perspective.

The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling.

We cloned the inebriated homologue MasIne from Manduca sexta and expressed it in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca(2+), as revealed by activation of the endogenous Ca(2+)-activated Cl(-) current. This Ca(2+) release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat gamma-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K(+) transport, which suggests that it may have a role in ion transport, particularly of K(+). We propose that stimulation of MasIne releases intracellular Ca(2+) in native tissues, activating Ca(2+)-dependent K(+) channels, and leading to K(+) transport.

Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds.

Fast synaptic transmission depends on the selective ionic permeability of transmitter-gated ion channels. Here we show changes in the ion selectivity of neuronal P2X transmitter-gated cation channels as a function of time (on the order of seconds) and previous ATP exposure. Heterologously expressed P2X2, P2X2/P2X3 and P2X4 channels as well as native neuronal P2X channels possess various combinations of mono- or biphasic responses and permeability changes, measured by NMDG+ and fluorescent dye. Furthermore, in P2X4 receptors, this ability to alter ion selectivity can be increased or decreased by altering an amino-acid residue thought to line the ion permeation pathway, identifying a region that governs this activity-dependent change.

H+ permeation and pH regulation at a mammalian serotonin transporter.

The rat serotonin transporter expressed in Xenopus oocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is approximately 10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H+/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H+ by reversal potential measurements. Similar H+-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na+/glucose transporter. The high conductance and high selectivity of the H+-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a "proton-wire mechanism") formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose-response relationships for these two effects suggest that two H+ binding sites, with pKa values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters.

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes.

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.

Cloning and expression of a rat brain GABA transporter.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.