Turkey coronaviral enteritis causes significant economic loss in the turkey industry around the world because of increased mortality and uneven flock growth. Neutralizing epitopes on the surface of the immunodominant spike (S) protein can be used as marker for detection of protective neutralizing antibodies and vaccine development. A protocol to identify a neutralizing epitope-containing segment in turkey coronavirus (TCoV) S protein is described in this chapter. Ten fragments covering the full length of S protein are expressed and purified to generate polyclonal antibodies for testing antigenicity by immunofluorescent antibody (IFA) assay and virus neutralization (VN) titer by VN assay. The carboxyl terminal region of the S1 protein (Mod4F/Epi4R) and the combined carboxyl terminal S1 and amino terminal S2 protein (4F/4R) were shown to contain the neutralizing epitopes. Neutralizing epitope-containing segment in TCoV S protein can be utilized for diagnostic serology to monitor the immune status of turkey flocks infected with TCoV or vaccinated with TCoV vaccines for the control of the disease.

Turkey coronavirus (TCoV) has been associated with poult enteritis and mortality syndrome (PEMS) since the 1990s and continued to cause significant economic loss in the turkey industry around the world in recent years. It is important to diagnose clinically TCoV-suspected turkeys showing ruffled feathers, wet droppings, and decreased body weight gain accurately by confirming the diagnosis with histopathology, immunofluorescence antibody (IFA) assay with anti-TCoV antiserum, reverse transcription PCR targeting TCoV genes, and sequencing. Infectious challenge of the newly isolated TCoV to the turkey poults is critical to determine the pathogenicity and serotypes of the new isolate by clinical signs, gross pathology, histopathology, and cross neutralization assay. A series of procedures to perform diagnostic investigation and confirmation of emerging turkey coronaviral enteritis is described in this chapter.

Turkey coronavirus (TCoV) is responsible for highly contagious enteritis in turkeys and causes significant loss in the turkey industries around the world. Spike (S) protein is the major structural protein of TCoV and inducer of protective antibodies. Segments containing neutralizing epitopes have been identified on TCoV S protein and used for vaccine development and diagnostic serology. A protocol to use recombinant S protein segment with neutralizing epitopes for enzyme-linked immunosorbent assay (ELISA) is described in this chapter. The carboxyl terminal region of the S1 protein (Mod4F/Epi4R) and the combined carboxyl terminal S1 and amino terminal S2 protein (4F/4R) are expressed by Escherichia coli and purified by His-Bind column chromatography. In addition to virus neutralization (VN) assay, antibody-capture ELISA based on neutralizing epitope-containing segment in TCoV S protein can be used to monitor the protective immune status of turkeys infected with TCoV or inoculated with the vaccines against TCoV.

The placenta is the primary source of serotonin (5-HT) for fetal development, programming fetal neural wiring in humans and other mammals. The fluctuation in maternal 5-HT affects fetal neurogenesis with life-long consequences, however, its mechanisms have not been well known. The chicken embryo, independent of mater-nal neurohormonal influence, may offer an ideal model for studying the mechanisms of prenatal 5-HT exposure altering postnatal physiological homeostasis and behavioral exhibition. To investigate the fine-tuning of 5-HT to the early embryonic neurodevelopment, 10 mg and 20 mg 5-HT were secretively injected to chicken embryos before incubation. 5-HT exposure mainly affected the neural development in the pons and midbrain, altered the serotoninergic and dopaminergic (DAergic) neuronal morphology, nucleus distribution, and their metabolisms and related gene expressions. The comprehensive effect of 5-HT exposure was not dosage-dependent but the working pathways differed, 10 mg 5-HT exposure reduced 5-HT turnover rate, increased 5-HT 1a receptor expres-sion, and facilitated the ventral tegmental area neuronal development; while 20 mg 5-HT exposure increased the serotoninergic and DAergic neurotransmission and enhanced serotoninergic regulation to the hypothalamus. These findings indicate that the 5-HT exposure effect can be achieved via different paths by modifying the embry-onic serotonergic (5-HTergic) and DAergic systems and altering fetal 5-HTergic influence on the thalamocortical circuit and hypothalamic-pituitary-adrenal axis. These results may offer a novel sight for understanding the func-tion of 5-HT during neurodevelopment and raise the possibility for using selective 5-HT reuptake inhibitors to reg-ulate emotional and mental wellness during early pregnancy and possible risks of complications for babies. (c) 2021 IBRO. Published by Elsevier Ltd. All rights reserved.

Avian species have immune system to fight invading pathogens. The immune system comprises innate and adaptive immunity. Innate immunity relies on pattern recognition receptors to sense particular molecules present in pathogens, i.e. pathogen-associated molecular patterns (PAMPs), or danger signals in the environment, i.e. danger-associated molecular patterns (DAMPs). Cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are the sensors recognizing cytoplasmic PAMP and/or DAMP. Among common avian species, chickens do not have RIG-I whereas ducks and finches do. Therefore, the other RLR member, melanoma differentiation-associated gene 5 (MDA5), is believed to play an important role to recognize intracellular pathogens in chickens. Chicken MDA5 has been identified and its function determined. Chicken MDA5 maintains the same domain architecture compared with MDA5 analogs in other animal species. The expression of chicken MDA5 was upregulated when a synthetic double-stranded RNA (dsRNA), polyriboinosinic:polyribocytidylic acids (poly(I:C)), was transfected into chicken cells, whereas that did not change when cells were incubated with poly(I:C). The enhanced expression of chicken MDA5 in chicken cells upregulated the expression of chicken interferon-[Formula: see text] (IFN-[Formula: see text]). The infection of dsRNA infectious bursal disease virus (IBDV) in non-immune cells triggered the activation of chicken MDA5 signaling pathway, leading to the production of IFN-[Formula: see text] and subsequent response of IFN-stimulated genes. Furthermore, in immune cells like macrophages, chicken MDA5 participated in sensing the infection of IBDV by activating downstream antiviral genes and molecules and modulating adaptive immunity.On the contrary, one of cytoplasmic NLR member, NLR family pyrin domain containing 3 (NLRP3), was cloned and functionally characterized in chicken cells. Chicken NLRP3 conserved the same domain architecture compared with NLRP3 analogs in other animal species. Chicken NLRP3 was highly expressed in kidney, bursa of Fabricius and spleen. The production of mature chicken interleukin 1 [Formula: see text] (IL-1[Formula: see text] in chicken macrophages was stimulated by lipopolysaccharide (LPS) treatment followed by short ATP exposure.In summary, chicken MDA5 was a cytoplasmic dsRNA sensor that mediated the production of type I IFN upon ligand engagement, whereas NLRP3 sensed danger signals, such as ATP, in the cytoplasm and cleaved pro-IL-1[Formula: see text] to produce mature IL-1[Formula: see text]. Chicken MDA5 was not only involved in the activation of innate immune responses in non-immune and immune cells, but it also participated in modulating adaptive immunity in immune cells. Chicken NLRP3 participated in the production of mature chicken IL-1[Formula: see text] upon ligand engagement.

Serotonin (5-HT) acts as a morphogen influencing embryonic brain development, and as a neurotransmitter regulating multiple biological functions with lifelong effects on animal physical, physiological and mental health, especially during the rapid growth phase prior to birth when embryos face many challenges to reach structural and functional completion. In this study, the development of the serotoninergic (5-HTergic) system and its modulatory effect on the dopaminergic (DAergic) system and related neural circuits were investigated during the mid-late embryogenesis, embryonic day (E)12-E20, in the chicken's brain. During 5-HTergic neuronal maturation, a growth-related anatomical and functional remodeling was highlighted: the 5-HT neurons continuously grew during E12-E20 except for a remarkable regression during E14-E16. Correspondingly, there was a time-dependent change in the 5-HT synthetic capacity. Specifically, 5-HT concentrations in the raphe nuclei increased from E12 to E14, reaching a first plateau during E14-E16, then continuously increased up to E19, and reaching a second plateau between E19-E20. The second plateau of the 5-HT concentration was in correspondence with the establishment of the 5-HTergic autoregulatory loop during E19-E20 and the development of the DAergic system. The DA concentrations remained unchanged from E12 to E16, then started to increase at E16, reaching a maximum at E19, and diminished before hatching. The unique developing time sequence between the 5-HTergic and DAergic systems suggests that the 5-HTergic system may play a critical role in forming the 5-HT - DA neural circuit during chicken embryogenesis. These results provide new insights for understanding the functional organization of the 5-HTergic system during embryonic development and raise the possibility that prenatally modulating the 5-HTergic system may lead to long-lasting brain structural and functional alterations.

Abstract Background Gastric wall edema has not been reported as a complication of acute pancreatitis in dogs. Objective To describe the ultrasonographic features of gastric wall thickening in dogs with acute pancreatitis. Animals Fourteen dogs with ultrasonographic evidence and clinical diagnosis of acute pancreatitis, with ultrasonographic evidence of increased gastric wall thickness (>5 mm). Methods A retrospective search in the medical records from 2014 to 2016 was performed to identify dogs that had ultrasonographic evidence of acute pancreatitis, that had increased thickness of the gastric wall and that were diagnosed with acute pancreatitis clinically. The gastric wall changes such as thickness, layering appearance, echogenicity, distribution of lesions, and perigastric changes were recorded. Serial ultrasonographic examination and histopathological findings were recorded if available. Results Mean gastric wall thickness was 9.9 ± 4.0 mm (SD). A complete loss of wall layering was observed in 2 dogs. Thickening of the submucosal layer was observed in 12 dogs, and 5 of them had concurrent muscularis layer thickening. The echogenicity of thickened submucosal layer was intermediate hyperechoic. Lacy appearances were present within the thickened submucosal layer in 7 dogs and in the muscularis layer of 1 dog. Thickening was focal in 12 dogs and adjacent to the diseased pancreas. Subsequent resolution of gastric wall thickening was observed in 3 dogs (range 3‐28 days) via follow‐up ultrasound. One dog underwent necropsy, and gastric wall edema was confirmed histopathologically. Conclusions and Clinical Importance Findings indicated that gastric wall thickening presumably because of edema could be a complication of acute pancreatitis.

Experimental infection of a high-passage turkey coronavirus passaged serially in embryonated turkey eggs for 344 times (P344 TCoV 540) showed no enteritis-related clinical signs, decreased body weight gains, gross, and microscopic lesions. TCoV spike (S) protein specific antibodies appeared from 14 days post infection (dpi) and increased gradually. Virus neutralization (VN) titers of the serum from P344 TCoV 540-inoculated turkeys were 1:13 at 14 dpi, 1:16 at 28 dpi, and 1:36 at 56 dpi against P344 TCoV 540. P344 TCoV 540-inoculated turkeys were protected against the challenge by homologous P344 TCoV 540 completely or low passage P3 TCoV 540 partially as revealed by lack of histopathological alterations, absence of TCoV by immunofluorescent antibody assay in the intestines, and reduction in TCoV viral RNA loads in the intestines and feces. The serum from P344 TCoV 540-vaccinated turkeys had higher VN titers against P344 TCoV 540 than those against P3 TCoV 540. P344 TCoV 540 had 52 amino acid substitutions as compared to those of P3 TCoV in the S protein. The results indicated that a high passage TCoV can induce protective humoral and cellular immune response and have potentials to become an attenuated vaccine.

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.

Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) continues to infect turkey flocks, resulting in significant economic loss. Determining and understanding genetic relationships among different TCoV isolates or strains is important for controlling the disease. Using two-step RT-PCR assays that amplify the full length of TCoV spike (S) gene, TCoV isolates can be sequenced, analyzed, and genotyped. Described in this chapter is the protocol on PCR amplification and sequencing analysis of full-length TCoV S gene. Such protocol is useful in molecular epidemiology for establishing an effective strategy to control the transmission of TCoV among turkey flocks.

Expression and purification of turkey coronavirus (TCoV) nucleocapsid (N) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kDa is induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressed N protein inclusion body is extracted and purified by chromatography on nickel-agarose column to near homogeneity. The protein recovery can be 10 mg from 100 ml of bacterial culture. The purified N protein is a superior source of TCoV antigen for antibody-capture ELISA for detection of antibodies to TCoV.

Turkey coronavirus (TCoV) infection induces the production of protective antibodies against the sequent exposure of TCoV. Serological tests to determine TCoV-specific antibodies are critical to evaluate previous exposure to TCoV in the turkey flocks and differentiate serotypes from different isolates or strains. A specific virus neutralization assay using embryonated turkey eggs and immunofluorescent antibody assay for determining TCoV-specific neutralizing antibodies is described in this chapter. Virus neutralization titer of turkey serum from turkeys infected with TCoV is the dilution of serum that can inhibit TCoV infection in 50 % of embryonated turkey eggs. Virus neutralization assay for TCoV is useful to monitor the immune status of turkey flocks infected with TCoV for the control of the disease.

Turkey coronavirus (TCoV) infection continues to threaten turkey industry. Because specific treatment and effective vaccination program are not available, rapid and cost-effective detection of antibodies to TCoV infection is an important control measure to monitor the disease status in the fields. Two antibody-capture enzyme-linked immunosorbent assay (ELISA) procedures for detection of antibodies to TCoV are outlined in this chapter. One ELISA method uses chicken infectious bronchitis coronavirus (IBV) as the coating antigen based on antigenic cross-reactivity between TCoV and IBV. The other method relies on a recombinant TCoV nucleocapsid protein. Both methods are useful for serological diagnosis of TCoV infection in the turkey flocks.

Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

The objective of the present study was to develop a multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis coronavirus (IBV), and bovine coronavirus (BCoV). Primers were designed from conserved or variable regions of nucleocapsid (N) or spike (S) protein gene among TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by the PCR reaction was used to amplify a portion of N or S gene of the corresponding coronaviruses. The PCR products were detected on agarose gel stained with ethidium bromide. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, were obtained for TCoV isolates. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene was obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene was obtained for BCoV. There were no PCR products with the same primers for Newcastle disease virus, Marek's disease virus, turkey pox virus, pigeon pox virus, fowl pox virus, reovirus, infectious bursal disease virus, enterovirus, astrovirus, Salmonella enterica, Escherichia coli, and Mycoplasma gallisepticum. Performance of the assay with serially diluted RNA demonstrated that the multiplex PCR could detect 4.8x10(-3) microg of TCoV RNA, 4.6x10(-4) microg of IBV RNA, and 8.0x10(-2) microg of BCoV RNA. These results indicated that the multiplex PCR as established in the present study is a rapid, sensitive, and specific method for differential detection of TCoV, IBV, and BCoV in a single PCR reaction.

This case report describes an episode of recurring severe necrotizing and haemorrhagic hepatitis and enteritis experienced in a flock of commercial layer pullets at 12 weeks of age and again at 18 weeks of age in Indiana. Pullets had been vaccinated at 10 weeks old using a trivalent Salmonella Enteritidis (SE)/Newcastle disease/infectious bronchitis oil-emulsion-inactivated vaccine. The pullets were found dead at 12 weeks with firm but friable, enlarged, haemorrhagic livers, enlarged spleens, and necrohaemorrhagic intestines. Histopathologic findings were consistent with a necrotizing and haemorrhagic enteritis and hepatitis. Livers had multiple intra-sinusoidal thrombi, intestines contained Gram-positive bacterial colonies, and spleens had marked lymphoid depletion. The pullets seemed to improve after antibiotic treatment. Pullets were vaccinated with an inactivated SE vaccine at 14 weeks of age. A second spike of mortality occurred at 18 weeks of age. Although clostridial enteritis and hepatitis were highly suspected in the two cases based on macroscopic and microscopic findings, no significant bacterial or viral agents were isolated from the livers and intestines. In summary, lesions in the liver and intestines are speculated to be due to repetitive vaccination, leading to an anamnestic response by the immune system, and resulting in an immune-mediated response. However, much of the pathogenesis is still unclear, and other causes such as unidentified infectious aetiology, transmissible amyloidosis, and hypersensitivity may need further investigation.

Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-β, IRF-1, IRF-10, IL-1β, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3, p53, glucose transporter-2 and glucose transporter-3, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. Taken together, the results indicate that the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport, except for granzyme K and CD8, was not differentially expressed in DNA-vaccinated chickens protected from IBDV challenge.

DNA vaccine coding for infectious bursal disease virus (IBDV) polyprotein gene and that for avian influenza virus (AIV) hemagglutinin (HA) gene have been shown to induce immunity and provide protection against the respective disease. The present study was carried out to determine whether an IBDV polyprotein gene-based DNA fused with AIV HA gene could trigger immune response to both IBDV and AIV. After transfection, VP2 and HA were detected in the cytoplasm and at cell membrane, respectively, by immunofluorescent antibody double staining method, suggesting the fusion strategy did not affect the location of protein expression. VP4 cleavage between VP2 and HA was confirmed by Western blot, indicating the fusion strategy did not affect VP4 function in transfected cells. After vaccination in chickens, the DNA construct VP24-HA/pcDNA induced ELISA and virus neutralizing antibodies against VP2 and hemagglutination inhibition antibody against the HA subtype. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination.

S / ISFM Congress 2015 EVOLUTION OF RETROVIRUS INFECTION RATE IN CATS AT THE VETERINARY HOSPITAL OF THE UNIVERSITY OF CHILE (1999–2014) Fernando G Elías, Loreto Muñoz Faculty of Veterinary Sciences, University of Chile,