Circulation and phylogenetic relationship of chicken and turkey-origin astroviruses detected in domestic ducks (Anas platyrhynchos domesticus).

The natural occurrence of chicken and turkey-origin astroviruses in domestic ducks (Anas platyrhynchos domesticus) is described. Twenty-two duck flocks were covered by this research. The liver, spleen, kidney and intestines were sampled and tested by reverse-transcription polymerase chain reaction for the presence of avian nephritis virus (ANV), chicken astrovirus (CAstV), turkey astrovirus (TAstV)-1, TAstV-2 and duck astrovirus. The astrovirus infection was confirmed in multiple organ samples from 59.1% of tested flocks. CAstV was detected in one flock, TAstV-2 in three flocks and ANV in 10 flocks. The molecular and phylogenetic analysis of the small open reading frame (ORF) 1b fragment (130 nucleotides) of all chicken and turkey-origin astroviruses detected in ducks showed that ANV-sequence group was more distant from CastV, TAstV-1 and TAstV-2 sequences, which formed a separate, more related group. ANV sequences were divided into three subgroups, suggesting that several types of ANV were circulating in Croatian duck flocks. The comparison of the partial ORF 1b (254 nucleotides) duck ANV sequences with 21 ANVs detected in various avian species (chickens, turkeys, geese, guinea fowl and pigeons) revealed they shared the higher nucleotide (95.6 to 97.2%) and amino acid (98.8 to 100%) identity with two ANV-2-like sequences from chickens (GA-SEP-A451-05 and GA-CK-SEP ANV-364-2005). Phylogenetic neighbour-joining tree analysis based on the same nucleotide alignment, and performed using the Jukes-Cantor method, clustered the compared sequences into three groups. All analysed duck ANV sequences showed a close phylogenetic relationship with chicken-origin ANVs. Additional work is required to determine the significance and pathogenicity of chicken and turkey-origin astroviruses in domestic ducks.

Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos.

The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsy's disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.

Detection and characterization of avian nephritis virus in ducklings.

The first evidence of avian nephritis virus (ANV) in ducks is described. A diagnostic investigation was performed on three duck farms in Croatia. Samples from dead-in-shell ducklings and ducklings aged 30 days were collected and prepared for molecular and histopathological examination. Intestinal and liver samples were tested by polymerase chain reaction (PCR) for the presence of ANV, duck enteritis virus, duck hepatitis virus 1 and Derzsy's disease virus. Multiple tissues were collected for histological examination and lesions were found to be confined to the kidney and intestine. Moderate focal interstitial and periglomerular mononuclear cell infiltrates (mostly lymphocytes and plasma cells) were detected in the kidney. The duodenum showed rather diffuse pericryptal mononuclear cell hyperplasia (lymphocytes) and fibroplasia. ANV was detected by PCR in all the intestinal samples, while no other viruses were found. Sequence comparisons of the portion of the open reading frame 1b encoding the RNA-dependent RNA polymerase gene confirmed that the virus detected and sequenced from ducklings shared high nucleotide and amino acid identities with ANV-1. Additional work is required to determine the clinicopathological significance of ANV infection in ducks.

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia.

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.

The influence of BPC 157 on nitric oxide agonist and antagonist induced lesions in broiler chicks.

We describe the effects of nitric oxide (NO) agonists and antagonists and the influence of a novel organoprotective pentadecapeptide BPC 157, on the development of pulmonary hypertension syndrome and tissue lesions in chicks. Acute toxicity, which includes single dose application of saline (1 mL intraperitoneally (i.p.)), BPC 157 (10 micrograms/kg bw), L-NAME (NO antagonist, doses 50, 100, 150 mg/kg bw) and L-arginine (NO agonist/100 mg/kg bw with their combination L-NAME + BPC 157; L-NAME + L-arginine) was investigated. In this experiment pathohistological examination of the spleen, heart, liver and lungs and hematological analysis was conducted. In the chronic toxicity experiment, the animals were treated daily for 5 weeks with L-NAME (10 mg/kg bw), L-arginine (100 mg/kg bw), BPC 157 (10 micrograms/kg bw) and their combinations (L-NAME + BPC 157; L-NAME + BPC 157; L-NAME + L-arginine) i.p. Seven animals from each group, including controls (saline 1 mL i.p.) were killed every week. Application of L-NAME caused pulmonary hypertension syndrome (PHS) in the treated chicks, which was prevented by the simultaneous application of L-arginine and BPC 157. Pathohistological examination of both acute and chronic toxicity revealed that L-NAME caused severe tissue damage (myocardial and hepatic cell necrosis, necrosis of the lymphoid cells in the spleen) while L-arginine provoked predominantly congestion, edema and hemorrhages in all organs. The effect of L-NAME was successfully inhibited by the application of L-arginine and BPC 157 but the latter substance did not cause any tissue or organ damage. Hematological analysis shows significant hemoglobin and leukocyte number decrease in the L-NAME-treated groups of chicks.

Pentadecapeptide BPC 157 positively affects both non-steroidal anti-inflammatory agent-induced gastrointestinal lesions and adjuvant arthritis in rats.

Besides a superior protection of the pentadecapeptide BPC 157 (an essential fragment of an organoprotective gastric juice peptide BPC) against different gastrointestinal and liver lesions, an acute anti-inflammatory and analgetic activity was also noted. Consequently, its effect on chronic inflammation lesions, such as adjuvant arthritis, and non-steroidal anti-inflammatory agents (NSAIAs)-induced gastrointestinal lesions was simultaneously studied in rats. In gastrointestinal lesions (indomethacin (30 mg/kg s.c.), aspirin (400 mg/kg i.g.) and diclofenac (125 mg/kg i.p.) studies, BPC 157 (10 micrograms or 10 ng/kg i.p.) was regularly given simultaneously and/or 1 h prior to drug application (indomethacin). In the adjuvant arthritis (tail-application of 0.2 mL of Freund's adjuvant) studies (14 days, 30 days, 1 year) BPC 157 (10 micrograms or 10 ng/kg i.p.), it was given as a single application (at 1 h either before or following the application of Freund's adjuvant) or in a once daily regimen (0-14th day, 14-30th day, 14th day-1 year). Given with the investigated NSAIAs, BPC 157 consistently reduced the otherwise prominent lesions in the stomach of the control rats, as well as the lesions in the small intestine in the indomethacin groups. In the adjuvant arthritis studies, the lesion's development seems to be considerably reduced after single pentadecapeptide medication, and even more attenuated in rats daily treated with BPC 157. As a therapy of already established adjuvant arthritis, its salutary effect consistently appeared already after 2 weeks of medication and it could be clearly seen also after 1 year of application. Taking together all these results, the data likely point to a special anti-inflammatory and mucosal integrity protective effect.

The effect of pentadecapeptide BPC 157, H2-blockers, omeprazole and sucralfate on new vessels and new granulation tissue formation.

A clear protection of the gastrointestinal tract and an evident anti-inflammatory effect were shown for a novel stomach pentadecapeptide BPC 157 (i.p./i.g.) in comparison with several reference standards in various ulcer models along with a protection of endothelium and particular interaction with the NO-system. Thus, we evaluated whether this pentadecapeptide along with other gastroprotective agents could affect angiogenesis and the healing process in vivo using a procedure initially described by Szabo and co-workers. In each rat, two sterile sponges (1 x 1 x 0.25 cm; V = 0.25 mL) with the same quantities of BPC 157 (10 ng x mL(-1), 10 microg x mL(-1), 50 microg x kg(-1)) or reference agents (cimetidine: 10, 100, 500 mg x mL(-1); ranitidine: 2.5, 25, 250 mg x mL(-1); famotidine: 10, 50, 100 mg x mL(-1); omeprazole: 10, 50, 100 mg x mL(-1); sucralfate: 1, 5, 10 mg x mL(-1) were implanted subcutaneously in the lumbar region. The sponges were removed after 3 or 7 d, fixed in formalin, and processed for histologic and histochemical evaluation and morphometry assessment. Compared with the control values, the number of newly formed endothelial spaces inside newly formed granulation tissue was markedly increased in all animals treated with BPC 157, cimetidine, ranitidine, famotidine, sucralfate and omeprazole, a consistent finding noted after either 3 or 7 d. Compared with control values, markedly more granulation tissue was noted in the rats in the groups of animals treated with BPC 157 (50 microg) and in the rats treated with sucralfate in all dosages used, euthanized after 3 d. In all groups treated with H2-blockers however, similar values to those of controls were noted. Thus, it could be concluded that an evident angiogenic property was consistently noted for the novel pentadecapeptide BPC 157, H2-blockers (cimetidine, famotidine and ranitidine) and omeprazole, besides the well known angiogenic effect of sucralfate. Furthermore, unlike H2-blockers and omeprazole, BPC 157 stimulates the formation of granulation tissue, suggesting a particular activity, similar to that previously noted for sucralfate.

The influence of a novel pentadecapeptide, BPC 157, on N(G)-nitro-L-arginine methylester and L-arginine effects on stomach mucosa integrity and blood pressure.

The known effects of a novel stomach pentadecapeptide BPC157 (10 microg or 10 ng/kg), namely its salutary activity against ethanol (96%, i.g.)-induced gastric lesions (simultaneously applied i.p.) and in blood pressure maintenance (given i.v.), were investigated in rats challenged with a combination of N(G)-nitro-L-arginine methylester (L-NAME) (5 mg/kg i.v.), a competitive inhibitor of endothelium nitric oxide (NO)-generation and NO precursor, L-arginine (200 mg/kg i.v.) (D-arginine was ineffective). In the gastric lesions assay, NO agents were given 5 min before ethanol injury and BPC 157 medication. Given alone, BPC157 had an antiulcer effect, as did L-arginine, but L-NAME had no effect. L-NAME completely abolished the effect of L-arginine, whereas it only attenuated the effect of BPC 157. After application of the combination of L-NAME + L-arginine, the BPC157 effect was additionally impaired. In blood pressure studies, compared with L-arginine, pentadecapeptide BPC 157 (without effect on basal normal values) had both a mimicking effect (impaired L-NAME-blood pressure increase, when applied prophylactically and decreased already raised L-NAME values, given at the time of the maximal L-NAME-blood pressure increase (i.e., 10 min after L-NAME)) and preventive activity (L-arginine-induced moderate blood pressure decrease was prevented by BPC 157 pretreatment). When BPC 157 was given 10 min after L-NAME + L-arginine combination, which still led to a blood pressure increase, its previously clear effect (noted in L-NAME treated rats) disappeared. In vitro, in gastric mucosa from rat stomach tissue homogenates, BPC 157, given in the same dose (100 microM) as L-arginine, induced a comparable generation of NO. But, BPC 157 effect could not be inhibited by L-NAME, even when L-NAME was given in a tenfold (100 versus 1000 microM) higher dose than that needed for inhibition of the L-arginine effect. NO synthesis was blunted when the pentadecapeptide BPC 157 and L-arginine were combined. In summary, BPC 157 could interfere with the effects of NO on both gastric mucosal integrity and blood pressure maintenance in a specific way, especially with L-arginine, having a more prominent and/or particularly different effect from that of NO.

Effects of histamine on gizzard erosions and on the activity of selected enzymes in chickens.

Dose- (5, 10 and 15 mg/kg body mass) and time-dependent (2, 6, 12 and 24 h after treatment) effects of histamine on gizzard lesions and serum AST, ALT and CK activities of chickens are reported. Morphometric results and histopathological examination revealed that the most effective histamine dose which induced severe gizzard lesions was 10 mg/kg b.m., especially 2, 6 and 12 h after administration. No difference from the control values was found after 24 h. That dose also induced an elevation of serum enzyme (AST, ALT, CK) activities, which was most expressed for the activity of ALT.

Stress induced gizzard erosion in chicks. I. Gross and histopathological findings.

A model of stress induced gizzard erosions is described. Three day old chickens were deprived of food for 24 h, and after that they were immersed in tap water (17 degrees C) for five seconds. Group A was sacrificed immediately before being subjected to stress, group B one hour, group C two hours and group D three hours after water immersion stress. Each group consisted of 10 animals. Gizzard lesions in group A were very mild and the main feature was discoloration of the gizzard lining. In groups B, C, and D the lesions were more severe and they were characterised by gizzard erosions, ulcerations and hemorrhages especially pronounced in group D. This model produced lesions in all stressed animals.

Gizzerosine-induced histopathological lesions in broiler chicks.

1. The aim of this study was to investigate pathomorphological changes in broiler chicks fed with different doses of gizzerosine, a substance produced during the heat treatments of fish meal. 2. The experiment was carried out in Ross broiler chicks which were divided into three groups: group A received 100% of non-medicated commercial mash for broiler chicks. During an experimental 5-d period, 50% of commercial mash was replaced with unheated fish meal (0.65 ppm gizzerosine) in group B and in group C with heated fish meal (1.15 ppm gizzerosine). Fourteen chicks from each group were killed every day. Samples of gastrointestinal and lymphoid organs, lung, pancreas, liver, brain and kidney tissue were sampled for histopathological analysis. Organs were embedded in paraffin and stained with hematoxylin-eosin stain and using periodic acid-Schiff reagent (PAS) and Sudan III (frozen sections). 3. Necropsy did not reveal notable differences between treated groups. There were no significant histopathological changes in immunocompetent organs nor in the lungs, the pancreas, the kidney or the brain. Sharply demarcated multiple vacuoles were observed in the myocardium in group C toward the end of the experiment. In group C, the prevalent changes in the gizzard and the proventriculus were slight to severe cuticle erosions and oedema of the lamina propria with or without multiple vacuoles, respectively, towards the end of the experiment. The most prominent changes toward the end of the experiment were dispersed cell vacuolisation in duodenal, jejunual, ileal and caecal lamina propria in group C. 4. In conclusion, it should be emphasised that extra-gizzard gizzerosine-induced lesions are probably not mediated by H2-receptor stimulation, but could be a consequence of cellular hypoxia.

Pentadecapeptide BPC 157 interactions with adrenergic and dopaminergic systems in mucosal protection in stress.

Since superior protection against different gastrointestinal and liver lesions and antiinflammatory and analgesic activities were noted for pentadecapeptide BPC (an essential fragment of an organoprotective gastric juice protein named BPC), the beneficial mechanism of BPC 157 and its likely interactions with other systems were studied. Hence its beneficial effects would be abolished by adrenal gland medullectomy, the influence of different agents affecting alpha, beta, and dopamine receptors on BPC 157 gastroprotection in 48 h restraint stress was further investigated. Animals were pretreated (1 hr before stress) with saline (controls) or BPC 157 (dissolved in saline) (10 microg or 10 ng/kg body wt intraperitoneally or intragastrically) applied either alone to establish basal conditions or, when manipulating the adrenergic or dopaminergic system, a simultaneous administration was carried out with various agents with specific effects on adrenergic or dopaminergic receptors [given in milligrams per kilogram intraperitoneally except for atenolol, which was given subcutaneously] phentolamine (10.0), prazosin (0.5), yohimbine (5.0), clonidine (0.1) (alpha-adrenergic domain), propranolol (1.0), atenolol (20.0) (beta-adrenergic domain), domperidone (5.0), and haloperidol (5.0) (peripheral/central dopamine system). Alternatively, agents stimulating adrenergic or dopaminergic systems--adrenaline (5.0) or bromocriptine (10.0)--were applied. A strong protection, noted following intragastric or intraperitoneal administration of BPC 157, was fully abolished by coadministration of phentolamine, clonidine, and haloperidol, and consistently not affected by prazosin, yohimbine, or domperidone. Atenolol abolished only intraperitoneal BPC 157 protection, whereas propranolol affected specifically intragastric BPC 157 protection. Interestingly, the severe course of lesion development obtained in basal conditions, unlike BPC 157 gastroprotection, was not influenced by the application of these agents. In other experiments, when adrenaline and bromocriptine were given simultaneously, a strong reduction of lesion development was noted. However, when applied separately, only adrenaline, not bromocriptine, has a protective effect. Thus, a complex protective interaction with both alpha-adrenergic (eg, catecholamine release) and dopaminergic (central) systems could be suggested for both intragastric and intraperitoneal BPC 157 administration. The involvement of beta-receptor stimulation in BPC 157 gastroprotection appears to be related to the route of BPC 157 administration. The demonstration that a combined stimulation of adrenergic and dopaminergic systems by simultaneous prophylactic application of adrenaline (alpha- and beta-receptor stimulant) and bromocriptine (dopamine receptor agonist) may significantly reduce restraint stress lesions development provides insight for further research on the beneficial mechanism of BPC 157.

Beneficial effect of a novel pentadecapeptide BPC 157 on gastric lesions induced by restraint stress, ethanol, indomethacin, and capsaicin neurotoxicity.

Very recently, the integrity of capsaicin somatosensory neurons and their protection were suggested to be related to the activity in nociception of a newly discovered 15-amino acid peptide, BPC 157, shown to have strong beneficial effect on intestinal and liver lesions. Therefore, from this viewpoint, we have studied the gastroprotective effect of the pentadecapeptide BPC 157, on gastric lesions produced in rats by 96% ethanol, restraint stress, and indomethacin. The possible involvement of sensory neurons in the salutary actions of BPC 157 (10 micrograms/kg, 10 ng/kg intraperitoneally) was studied with capsaicin, which has differential effects on sensory neurons: a high dose in adult (125 mg/kg subcutaneously, 3 months old) or administration (50 mg/kg subcutaneously) to neonatal animals (age of the 7 days) destroys sensory fibers, whereas a low dose (500 micrograms/kg intraperitoneally) activates neurotransmitter release and protective effects on the mucosa. In the absence of capsaicin, BPC 157 protected gastric mucosa against ethanol, restraint, and indomethacin application. In the presence of neurotoxic doses of capsaicin, the negative influence of capsaicin on restraint, ethanol, or indomethacin lesions consistently affected salutary activity of BPC 157. However, BPC 157 protection was still evident in the capsaicin-treated rats (either treated as adults or as newborns) in all of these assays. Interestingly, after neonatal capsaicin treatment, a complete abolition of BPC gastroprotection was noted if BPC 157 was applied as a single nanogram-regimen, but the mucosal protection was fully reversed when the same dose was used daily. In line with the excitatory dose of capsaicin the beneficial effectiveness of BPC 157 appears to be increased as well. Taken together, these data provide evidence for complex synergistic interaction between the beneficial effectiveness of BPC 157 and peptidergic sensory afferent neuron activity.