Janus kinase/signal transducer and activator of transcription signaling pathway-related genes STAT3, SOCS3 and their role in thiram induced tibial dyschondroplasia chickens.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg-1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage.

Altered expression of lactate dehydrogenase and monocarboxylate transporter involved in lactate metabolism in broiler wooden breast.

Wooden breast (WB) results in significant losses to the broiler industry due to reductions in meat quality. While the etiology of WB is unknown, it is believed to be associated with localized hypoxia and decreased lactate levels in skeletal muscles, indicating the presence of altered lactate metabolism in WB. We hypothesized that the expression levels of the major signaling molecules that control lactate metabolism, including lactate dehydrogenases (LDHA and LDHB) and monocarboxylate transporters (MCT1 and MCT4), were altered in WB. Therefore, the objectives of this study were to evaluate whether there were changes in mRNA and protein levels of LDHA, LDHB, MCT1, and MCT4 in WB compared to normal breast (NB) muscles. Biochemical analysis for LDH enzyme activity in NB and WB muscles was studied. MicroRNA375 (miR-375) expression, known to be inversely associated with LDHB protein expression in human cells, was also investigated. The level of LDHA mRNA was 1.7-fold lower in WB tissues than in NB tissues (P < 0.0001). However, the LDHA protein levels were similar in WB and NB tissues. In contrast, the levels of LDHB mRNA and protein were 8.4-fold higher (P < 0.002) and 13.6-fold higher (P < 0.02) in WB than in NB tissues, respectively. The level of miR-375 was not different between WB and NB muscles. The specific LDH isoenzyme activity that converted lactate to pyruvate was 1.8-fold lower in WB compared to NB tissues (P < 0.01). The level of MCT1 mRNA was 2.3-fold higher in WB than those in NB muscles (P < 0.02). However, this upregulation was not observed with MCT1 protein expression levels. The expression levels of MCT4 mRNA and protein were elevated 2.8-fold (P < 0.02) and 3.5-fold (P < 0.004) in WB compared to NB tissues, respectively. Our current findings suggest the potential roles of LDHB and MCT4 on lactate metabolism and provide a unique molecular elucidation for altered lactate homeostasis in WB muscles of broilers.

Gga-miR-30d regulates infectious bronchitis virus infection by targeting USP47 in HD11 cells.

Avian infectious bronchitis virus (IBV) is a coronavirus which infects chickens and causes severe economic losses to the poultry industry worldwide. MicroRNAs (miRNAs) are important intracellular regulators and play a pivotal role in viral infections. In previous studies, we have revealed that IBV infection caused a significant down-regulation of gga-miR-30d expression in chicken kidneys. In present study, we investigated the role of gga-miR-30d in the process of IBV infection of HD11 cell line in vitro. By transfecting the mimics and inhibitor of gga-miR-30d, it was found that overexpressed gga-miR-30d inhibited IBV replication. Contrarily, low-expressed gga-miR-30d promoted IBV replication. In addition, dual-luciferase reporter assays revealed that ubiquitin-specific protease 47 (USP47), a deubiquitinase-encoding gene, was a target for gga-miR-30d. This is the first study demonstrating that miRNAs regulate IBV replication by regulating the deubiquitinating enzyme (DUBs).

Effect of challenge dose of plasmid-mediated extended-spectrum ß-lactamase and AmpC ß-lactamase producing Escherichia coli on time-until-colonization and level of excretion in young broilers.

Plasmid-mediated extended-spectrum ß-lactamase and AmpC ß-lactamase (ESBL/pAmpC) producing bacteria are present at all levels of the broiler production pyramid. Young birds can be found positive for ESBL/pAmpC-producing Escherichia coli shortly after arrival at farm. The aim of this study was to determine the effect of different challenge doses of ESBL/pAmpC-producing E. coli on time-until-colonization and the level of excretion in young broilers. One-day-old broilers (specific-pathogen free (SPF) and conventional Ross 308) were housed in isolators and challenged with 0.5 ml ESBL/pAmpC-producing E. coli strains of varying doses (101-105 CFU/ml). Presence and concentration (CFU/gram feces) of ESBL/pAmpC-producing E. coli and total E. coli were determined longitudinally from cloacal swabs, and in cecal content 72 h after challenge. Higher challenge doses resulted in shorter time-until-colonization. However, even the lowest dose (101 CFU/ml) resulted in colonization of the broilers which excreted >106 CFU/gram feces 72 h after inoculation. Conventional broilers were colonized later than SPF broilers, although within 72 h after challenge all broilers were excreting ESBL/pAmpC-producing E. coli. A probabilistic model was used to estimate the probability of colonization by initial inoculation or transmission. The higher the dose the higher the probability of excreting ESBL/pAmpC-producing E. coli as a result of inoculation. In conclusion, low initial doses of ESBL/pAmpC-producing E. coli can result in rapid colonization of a flock. Interventions should thus be aimed to eliminate ESBL/pAmpC-producing bacteria in the environment of the hatchlings and measures focusing at reducing colonization and transmission of ESBL/pAmpC-producing E. coli should be applied shortly after hatching.

Plastrum Testudinis Extract Mitigates Thiram Toxicity in Broilers via Regulating PI3K/AKT Signaling.

Tibial dyschondroplasia (TD) negatively affects broilers all over the world, in which the accretion of the growth plate (GP) develops into tibial proximal metaphysis. Plastrum testudinis extract (PTE) is renowned as a powerful antioxidant, anti-inflammatory, and bone healing agent. The current study was conducted to evaluate the efficacy of PTE for the treatment of thiram-induced TD chickens. Broilers (day old; n = 300) were raised for 3 days with normal feed. On the 4th day, three groups (n = 100 each) were sorted, namely, the control (normal diet), TD, and PTE groups (normal diet+ thiram 50 mg/kg). On the 7th day, thiram was stopped in the TD and PTE group, and the PTE group received a normal diet and PTE (30 mg/kg/day). Plastrum testudinis extract significantly restored (p < 0.05) the liver antioxidant enzymes, inflammatory cytokines, serum biochemicals, GP width, and tibia weight as compared to the TD group. The PTE administration significantly increased (p < 0.05) growth performance, vascularization, AKT (serine/threonine-protein kinase), and PI3K expressions and the number of hepatocytes and chondrocytes with intact nuclei were enhanced. In conclusion, PTE has the potential to heal TD lesions and act as an antioxidant and anti-inflammatory drug in chickens exposed to thiram via the upregulation of AKT and PI3K expressions.

Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18.

Previous research revealed the induction of chicken USP18 (chUSP18) in the lungs of chickens infected with highly pathogenic avian influenza viruses (HPAIVs). This activity was correlated with the degree of pathogenicity of the viruses to chickens. As mammalian ubiquitin-specific protease (USP18) is known to remove type I interferon (IFN I)-inducible ubiquitin-like molecules from conjugated proteins and block IFN I signalling, we explored the function of the chicken homologue of USP18 during avian influenza virus infection. With this aim, we cloned chUSP18 from cultured chicken cells and revealed that the putative chUSP18 ORF comprises 1137 bp. Comparative analysis of the predicted aa sequence of chUSP18 with those of human and mouse USP18 revealed relatively high sequence similarity among the sequences, including domains specific for the ubiquitin-specific processing protease family. Furthermore, we found that chUSP18 expression was induced by chicken IFN I, as observed for mammalian USP18. Experiments based on chUSP18 over-expression and depletion demonstrated that chUSP18 significantly enhanced the replication of a low-pathogenic avian influenza virus (LPAIV), but not an HPAIV. Our findings suggest that chUSP18, being similar to mammalian USP18, acts as a pro-viral factor during LPAIV replication in vitro.

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. IMPORTANCE: Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

Ectonucleotidases and adenosine deaminase activity in laying hens naturally infected by Salmonella Gallinarum and their effects on the pathogenesis of the disease.

Salmonella Gallinarum is the etiologic agent of fowl typhoid that affects chickens and turkeys causing egg production drops, infertility, lower hatchability, high mortality, and as a consequence severe economic losses to the poultry industry. The alterations in NTPDase and adenosine deaminase (ADA) activities have been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this infection. Thus, the aim of this study was to evaluate the activities of NTPDase, 5'nucleotidase, and ADA in serum and hepatic tissue of laying hens naturally infected by Salmonella Gallinarum. Liver and serum samples were collected of 27 laying hens (20 S. Gallinarum infected and 7 uninfected). NTPDase and 5'-nucleotidase activities in serum were increased (P < 0.001) in infected animals to hydrolysis of substrate ATP, ADP and AMP. In addition, it was observed decreased (P < 0.001) in ADA activity in serum of laying hens naturally infected by S. Gallinarum; as well as increased (P < 0.001) ADA activity in liver tissue of infected laying hens. Histopathological analyses revealed that S. Gallinarum caused fibrinoid necrosis in liver and spleen associated with infiltrates of heterophils, macrophages, lymphocytes, and plasma cells. Considering that NTPDase and ADA are involved in the cell-mediated immunity, this study suggests that activities of these enzymes could be important biomarkers to determine the severity of inflammatory and immune responses in salmonellosis, contributing to clarify the pathogenesis of the disease.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages.

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 10(7) plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-ß (IFN-ß) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2', 5'-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1ß (IL-1ß) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 µM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-ß and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would diminish intracellular pools of nutrients and inhibit pathogen replication.

Structural view and substrate specificity of papain-like protease from avian infectious bronchitis virus.

Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the ß-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.

Impacts of the feed contaminant deoxynivalenol on the intestine of monogastric animals: poultry and swine.

Deoxynivalenol (DON) is one of the most prevalent cereal contaminants with major public health concerns owing to its high toxigenic potentials. Once ingested, DON first and foremost targets epithelial cells of the gastrointestinal tract, whose proper functioning, as the first line of defence, is of paramount importance for the host's health. Emerging evidences, summarized in this article, suggest that DON produces its toxicity primarily via activation of the mitogen-activated protein kinases (MAPKs) signalling pathway and alteration in the expression of genes responsible for key physiological and immunological functions of the intestinal tissue of chickens and pigs. The activation of MAPKs signalling cascade results in disruption of the gut barrier function and an increase in the permeability by reducing expression of the tight junction proteins. Exposure to DON also down-regulates the expression of multiple transporter systems in the enterocytes with subsequent impairment of the absorption of key nutrients. Other major intestinal cytotoxic effects of DON described herein are modulation of mucosal immune responses, leading to immunosupression or stimulation of local immune cells and cytokine release, and also facilitation of the persistence of intestinal pathogens in the gut. Both of the last events potentiate enteric infections and local inflammation in pigs and poultry, rendering enterocytes and the host more vulnerable to luminal toxic compounds. This review highlights the cytotoxic risks associated with the intake of even low levels of DON and also identifies gaps of knowledge that need to be addressed by future research.

A novel activation mechanism of avian influenza virus H9N2 by furin.

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA(1) allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.

Proteolytic activity alterations resulting from force-feeding in Muscovy and Pekin ducks.

We investigated liver protease activity in force-fed and non-force-fed ducks using zymography gels to better understand mechanisms underlying liver steatosis in palmipeds. Male Muscovy and Pekin ducks were slaughtered before and after a short period (13 d) while they were conventionally fed or force fed. The force-fed regimen contained a high level of carbohydrates and was delivered in large doses. Main hepatic proteases (matrix metalloprotease-2, calpains, and cathepsins) were extracted from raw liver and specifically activated within electrophoretic gels. Both force-fed Muscovy and Pekin ducks presented higher liver weights and BW associated with lower matrix metalloprotease-2 and m-calpain hepatic activities. On the other hand, hepatic cathepsin activity was not affected by force feeding. It was concluded that Muscovy and Pekin duck hepatic proteases are affected similarly by the force feeding. Thus, this cannot explain differences observed between Muscovy and Pekin ducks regarding their ability to develop hepatic steatosis generally reported in literature.

Isolation and characterization of a low pathogenic duck hepatitis A virus 3 from South Korea.

The D11-JW-018 strain of duck hepatitis A virus (DHAV) was isolated from 7-day-old ducklings in Kyeonggi province, South Korea with no clinical signs of typical hepatitis. Phylogenetic analyzed of whole genome showed that D11-JW-018 strain was belonged to DHAV-3 genotype. The pathogenicity of the D11-JW-018 strain in 1-, 7-, 14-, and 21-day-old ducklings was examined. Mortality of D11-JW-018 strain was lower than DRL-62 (DHAV-1) age-dependent but incubation period was longer in 1-day-old ducklings. Unlike DRL-62 strain infection, D11-JW-018 strain induced only liver discoloration without hemorrhagic mottling and lymphocyte infiltration and bile duct hyperplasia in histological lesion. The D11-JW-018 strain was detected only in the heart, liver, spleen, gall bladder, pancreas, and kidney among 12 organs in infected 1-day-old ducklings. Serum biochemical analyses revealed a significant difference in aspartate transaminase and alanine transaminase between the D11-JW-018 strain-infected ducklings and those infected with the DRL-62 strain (P<0.05). We identified the D11-JW-018 strain in South Korean ducklings and provide the characteristics of DHAV-3.

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose.

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5'/3'-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.

Ornithobacterium rhinotracheale has neuraminidase activity causing desialylation of chicken and turkey serum and tracheal mucus glycoproteins.

Neuraminidases (sialidases) are virulence factors of several poultry pathogens. Ornithobacterium rhinotracheale is a well known poultry pathogen causing respiratory disease in chickens and turkeys all over the world. We investigated whether O. rhinotracheale has neuraminidase enzymatic activity (NEAC). We tested NEAC in 47 O. rhinotracheale strains isolated from turkeys and chickens in eight countries. All strains showed relatively strong NEAC and considerable levels of NEAC were detected also in "cell-free supernatants" of their pelleted cells. Zymography using neuraminidase-specific chromogenic substrate indicated that a protein with molecular mass of ~40kDa and isoelectric point (pI) of ~8.0 is a putative neuraminidase of O. rhinotracheale. Notably, the genome of the type strain of O. rhinotracheale, DSM 15997 contains a gene (Ornrh_1957) encoding a putative neuraminidase with such Mw (39.5 kDa) and pI (8.5). We sequenced a corresponding genomic region of 20 O. rhinotracheale strains and found five distinct types of the neuraminidase gene (termed nanO) sequences. Most diversified nanO sequence was found in two strains isolated from chickens in Hungary in 1995. Their nanO sequences differ from that of the type strain (LMG 9086(T)) in 27 nucleotides. O. rhinotracheale neuraminidase showed capacity to cleave sialic acid from chicken and turkey glycoproteins. It cleaved sialic acid from SAα(2-6)gal moiety of their serum proteins, including immunoglobulin G (IgG) and transferrin. O. rhinotracheale also desialylated chicken and turkey tracheal mucus glycoprotens with SAα(2-3)gal moieties. This study provides the first evidence that O. rhinotracheale has neuraminidase which can desialylate glycoproteins of its natural hosts.

Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens.

We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.