AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

Prevalence and risk factors of hepatic lipid changes in bearded dragons (Pogona vitticeps).

Hepatic lipidosis is a common disease of captive bearded dragons (Pogona vitticeps). Diagnosis, prevention, and treatment of this condition are challenging, as there is minimal information in the literature. Our study determined the prevalence and epidemiological risk factors associated with the grade and severity of hepatic lipid changes in bearded dragons submitted for necropsy in 2 North American institutions. A total of 571 postmortem cases were retrieved, and from each pathology report the demographic data (age, sex) and the list of final diagnoses were extracted. For each case diagnosed with hepatic lipidosis, the archived sections of liver were reviewed and the severity of lipid change was stratified using a standardized histologic grading system. Descriptive statistics were used to estimate the prevalence of each grade and severity class. Associations between grade and severity, as well as demographic data and concurrent diseases, were explored using ordinal logistic regression analysis. On multiple logistic models, the occurrence of infectious disease and neoplasia was associated with decreased grade and severity of hepatic lipid changes, while the female sex and adult age were associated with an increased grade and severity. None of the other variables were significantly associated with hepatic lipid changes. These results suggest that reproductively active females and adult bearded dragons are predisposed to increasing hepatic lipid changes, while those with an underlying disease process have reduced hepatic lipid accumulation and changes, possibly due to increased fat catabolism. Data in this study can serve to benchmark the prevalence of hepatic lipidosis in bearded dragons and allow further investigations.

Morphological features of hepatic lipid changes in bearded dragons (Pogona vitticeps), and a proposed grading system.

Hepatic lipidosis is commonly diagnosed in pet bearded dragons (Pogona vitticeps). However, there are no studies detailing the histological features of hepatic lipid changes in this species. This study describes the microscopic features of lipid change and details an associated scoring system. Histologic hepatic sections were retrospectively evaluated from 252 bearded dragons submitted for necropsy. Pathologic assessment was used to develop a grading scheme with 2 qualitative, 1 quantitative, and 6 semi-quantitative microscopic parameters, which were refined based on variability. The final grading system developed for diffuse and panlobular lipid accumulation included 2 semi-quantitative and 1 quantitative categories: percentage of hepatocellular vacuolation, fibrosis, and hepatocellular swelling, respectively. Hepatocellular swelling was indirectly quantified by counting the number of nuclei per unit area. There was a strong positive correlation (P < .001) between the percentage of hepatocellular vacuolation and lipid content, a strong negative correlation (P < .001) between nuclear count and lipid content, and a moderate correlation (P < .001) between fibrosis and lipid content. Each category was given a numerical value ranging from 0 to 4, with the sum of each representing the final grade. Cutoff values stratified microscopic changes into mild (final grade 1-4), moderate (5-7), and severe (≥8). There was strong interrater agreement for assessment of vacuolization, fibrosis, and severity classification and moderate for hepatocellular swelling. This study documents the features of hepatic lipid changes in bearded dragons. Although a cutoff to differentiate pathologic from nonpathologic lipid accumulation could not be estimated, the proposed grading scheme can be used to inform future studies.

Congenital glaucoma in a tiger (Panthera tigris).

OBJECTIVE: To describe a case of congenital glaucoma in atiger (Panthera tigris). ANIMAL STUDIED: An 8-month-old intact female tiger wasreferred for suspected glaucoma of the right eye. The right eye was buphthalmicwith moderate episcleral injection, circumferential superficial cornealneovascularization, moderate corneal edema, and a fixed dilated pupil. Tapetalreflection was absent due to a mature cataract. Rebound tonometry under generalanesthesia revealed 70 mmHg and 21 mmHg in the right and left eye, respectively. PROCEDURE: A trans-conjunctival enucleation was performedand the globe was submitted for histopathology. RESULTS: Histopathology revealed a thin sclera, amorphousmaterial contouring an imperforate and hypoplastic iridocorneal angle, ahypoplastic lens with severe anterior-posterior compression, subcapsularepithelial hyperplasia, and Morganian globules, and segmental moderate retinalatrophy. Periodic acid-Schiff stain highlighted segmental dilations of theDescemet's membrane. Masson trichrome stain highlighted a pre-irido collagenmembrane. CONCLUSION: The tiger's age and histopathologic findingsare consistent with congenital goniodysgenesis. This is the first known reportof congenital glaucoma in a tiger.

Adeno-associated virus mediated expression of monoclonal antibody MR191 protects mice against Marburg virus and provides long-term expression in sheep.

Vectored monoclonal antibody (mAb) expression mediated by adeno-associated virus (AAV) gene delivery leads to sustained therapeutic mAb expression and protection against a wide range of infectious diseases in both small and large animal models, including nonhuman primates. Using our rationally engineered AAV6 triple mutant capsid, termed AAV6.2FF, we demonstrate rapid and robust expression of two potent human antibodies against Marburg virus, MR78 and MR191, following intramuscular (IM) administration. IM injection of mice with 1 × 1011 vector genomes (vg) of AAV6.2FF-MR78 and AAV6.2FF-MR191 resulted in serum concentrations of approximately 141 µg/mL and 195 µg/mL of human IgG, respectively, within the first four weeks. Mice receiving 1 × 1011 vg (high) and 1 × 1010 vg (medium) doses of AAV6.2FF-MR191 were completely protected against lethal Marburg virus challenge. No sex-based differences in serum human IgG concentrations were observed; however, administering the AAV-mAb over multiple injection sites significantly increased serum human IgG concentrations. IM administration of three two-week-old lambs with 5 × 1012 vg/kg of AAV6.2FF-MR191 resulted in serum human IgG expression that was sustained for more than 460 days, concomitant with low levels of anti-capsid and anti-drug antibodies. AAV-mAb expression is a viable method for prolonging the therapeutic effect of recombinant mAbs and represents a potential alternative "vaccine" strategy for those with compromised immune systems or in possible outbreak response scenarios.

Lymphoma in Psittacine Birds: A Histological and Immunohistochemical Assessment.

In psittacine birds, round cell neoplasms that originate from lymphocytes, plasma cells, histiocytes, or mast cells are sporadic and poorly described. The lack of morphological and immunohistochemical diagnostic criteria or grading schemes make specific diagnoses and prognoses challenging. We assessed cases of psittacine birds diagnosed with round cell neoplasia from 3 North American veterinary diagnostic laboratories to describe the diagnostic features of these tumors. For all cases, demographic data, anatomic distribution, histological features, and immunoreactivity for T (CD3) and B (Pax5 and MUM-1) cell markers were assessed using tissue microarrays and whole slide mounts. Thirty-eight psittacine birds representing 14 species were included. Tumors were mainly infiltrative and multicentric, were composed of homogenous sheets of round to polygonal cells, and commonly presented with a high mitotic count (average 21 mitoses per high-power field). Based on Pax5 immunoreactivity, B-cell lymphoma was most common (19/38 [50%]), and was significantly associated with involvement of the gastrointestinal and urogenital systems. Of the 38 cases, 6 (16%) were consistent with T-cell lymphoma, 3 (8%) with plasma cell tumor, and 3 (8%) were double-reactive for both B- and T-lymphocyte markers. This is the first study to describe morphologic and immunohistochemical features of round cell neoplasia in a large number of psittacine birds, and provides benchmark data for future studies aimed at elucidating the diagnosis and prognosis of these neoplasms. These data also provide useful information about reactivity of commercially available antibodies as lymphocyte markers in tissues of multiple psittacine species.

Iridociliary adenoma in a greater sulfur-crested cockatoo (Cacatua galerita galerita).

A 34-year-old female greater sulfur-crested cockatoo (Cacatua galerita galerita) was referred for suspected left globe rupture. Ophthalmic examination revealed effacement of the anterior chamber and cornea by a large mass. The left eye was enucleated due to suspicion of globe rupture, secondary to a neoplastic process or chronic trauma. Histopathological examination revealed complete effacement of the internal ocular structures by a neoplasm morphologically consistent with an iridociliary adenoma. The diagnosis was confirmed by Periodic acid-Schiff histochemistry and immunohistochemistry for S100, Melan-A/PNL2, and vimentin antigens. The cockatoo recovered well from surgery, with appropriate healing of the enucleation site, and no evidence of recurrence at 1-year follow-up. Key clinical message: To the authors' knowledge, this is the first report of iridociliary adenoma in a greater sulfur-crested cockatoo, and the third report of such a neoplasm in a psittacine species with a description of the use of immunohistochemistry to confirm a diagnosis of a rare tumor in a bird species.

Adénome iridociliaire chez un cacatoès à crête de soufre (Cacatua galerita galerita) . Une femelle cacatoès à crête de soufre (Cacatua galerita galerita) âgée de 34 ans a été référée pour suspicion de rupture du globe oculaire gauche. L'examen ophtalmique a révélé un effacement de la chambre antérieure et de la cornée par une masse importante. L'oeil gauche fut énucléé en raison d'une suspicion de rupture du globe oculaire, secondaire à un processus néoplasique ou à un traumatisme chronique. L'examen histopathologique a révélé un effacement complet des structures oculaires internes par un néoplasme morphologiquement compatible avec un adénome iridociliaire. Le diagnostic a été confirmé par histochimie avec coloration PAS (Periodic acid-Schiff ) et immunohistochimie pour les antigènes S100, Melan-A/PNL2 et vimentine. Le cacatoès s'est bien rétabli de la chirurgie, avec une guérison appropriée du site d'énucléation, et aucun signe de récidive à 1 an de suivi.Message clinique clé :À la connaissance des auteurs, il s'agit du premier rapport d'adénome iridociliaire chez un grand cacatoès à crête de soufre et du troisième rapport d'un tel néoplasme chez une espèce psittacidé avec une description de l'utilisation de l'immunohistochimie pour confirmer le diagnostic d'une tumeur rare chez une espèce d'oiseau.(Traduit par Dr Serge Messier).

Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines.

BACKGROUND: Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation. METHODS: The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID50) assay, respectively. RESULTS: The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 105 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre. CONCLUSIONS: This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.

Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica isolates: a two-year prospective study of small poultry flocks in Ontario, Canada.

BACKGROUND: Although keeping small poultry flocks is increasingly popular in Ontario, information on the antimicrobial susceptibility of enteric bacteria of such flocks is lacking. The current study was conducted on small poultry flocks in Ontario between October 2015 and September 2017, and samples were submitted on a voluntary basis to Ontario's Animal Health Laboratory. From each submission, a pooled cecal sample was obtained from all the birds of the same species from the same flock and tested for the presence of two common enteric pathogens, E. coli and Salmonella. Three different isolates from each E. coli-positive sample and one isolate from each Salmonella-positive sample were selected and tested for susceptibility to 14 antimicrobials using a broth microdilution technique. RESULTS: A total of 433 fecal E. coli isolates (358 chicken, 27 turkey, 24 duck, and 24 game bird) and 5 Salmonella isolates (3 chicken, 1 turkey, and 1 duck) were recovered. One hundred and sixty-seven chicken, 5 turkey, 14 duck, and 15 game bird E. coli isolates were pan-susceptible. For E. coli, a moderate to high proportion of isolates were resistant to tetracycline (43% chicken, 81% turkey, 42% duck, and 38% game bird isolates), streptomycin (29% chicken, 37% turkey, and 33% game bird isolates), sulfonamides (17% chicken, 37% turkey, and 21% duck isolates), and ampicillin (16% chicken and 41% turkey isolates). Multidrug resistance was found in 37% of turkey, 20% of chicken, 13% of duck, and 8% of game bird E. coli isolates. Salmonella isolates were most frequently resistant to streptomycin, tetracycline, and sulfonamides. Resistance to cephalosporins, carbapenems, macrolides, and quinolones was infrequent in both E. coli and Salmonella isolates. Cluster and correlation analyses identified streptomycin-tetracycline-sulfisoxazole-trimethoprim-sulfamethoxazole as the most common resistance pattern in chicken E. coli isolates. Turkey E. coli isolates compared to all the other poultry species had higher odds of resistance to tetracycline and ampicillin, and a higher multidrug resistance rate. CONCLUSIONS: Escherichia coli isolates were frequently resistant to antimicrobials commonly used to treat poultry bacterial infections, which highlights the necessity of judicious antimicrobial use to limit the emergence of multidrug resistant bacteria.

Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia.

BACKGROUND: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Lipid-Related Lesions in Quaker Parrots ( Myiopsitta monachus).

The Quaker parrot has been used as a psittacine model to study clinical lipidology and lipid-related disorders. However, while Quaker parrots appear to be anecdotally susceptible to a variety of spontaneous dyslipidemic disorders and lesions caused by excess lipid accumulation, epidemiologic data are lacking. A multicenter retrospective study on 652 pathology submissions (411 necropsies and 243 biopsies) from Quaker parrots was performed by recording the final pathological diagnoses, age, and sex for each bird. The prevalence of lesions associated with lipid metabolism, such as hepatic lipidosis, atherosclerosis, xanthomas, adipose tumors, coelomic steatitis/steatonecrosis, endogenous lipid pneumonia, and acute pancreatic necrosis/pancreatitis, was reported. Multiple logistic regression models were used to characterize the effects of sex and age on these lesions, and the prevalence of hepatic lipidosis and atherosclerosis was compared to those in a random sample of control psittacine birds. The raw prevalence of atherosclerosis and hepatic lipidosis was 5.6% (95% confidence interval [CI], 3.4%-7.8%) and 21.2% (95% CI, 17.2%-25.1%), respectively. While the prevalence of atherosclerosis was similar to other psittacine species, hepatic lipidosis was more common in Quaker parrots. Quaker parrots also showed a unique susceptibility to acute pancreatic necrosis with a prevalence of 12.9% (95% CI, 9.7%-16.1%). Male parrots were found to be more susceptible than females to lipid accumulation lesions ( P = .0024), including atherosclerosis ( P = .018) and hepatic lipidosis ( P < .001). This retrospective study confirms the high susceptibility of Quaker parrots to lipid-related disorders and presents epidemiological data that may be useful to avian clinicians, pathologists, and researchers using Quaker parrots.

Newcastle Disease Virus Infection in Quail.

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail ( Coturnix japonica) were experimentally inoculated with 108 EID50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail ( N2, N23) and 2 from chickens ( Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

BACKGROUND: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage. RESULTS: Results showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs. CONCLUSIONS: Results demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.

Vaccination of chickens decreased Newcastle disease virus contamination in eggs.

Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens.

Role of poultry in the spread of novel H7N9 influenza virus in China.

UNLABELLED: The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 10(6) 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE: The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

BACKGROUND: In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). METHODS: To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. RESULTS: At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (10(3.46) EID50 /0.1 ml) and drastically decreased at day 5 pi (10(0.9) EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 10(5.35) EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. CONCLUSIONS: Increased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4-5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus.

Delayed Newcastle disease virus replication using RNA interference to target the nucleoprotein.

Each year millions of chickens die from Newcastle disease virus (NDV) worldwide leading to severe economic and food losses. Current vaccination campaigns have limitations especially in developing countries, due to elevated costs, need of trained personnel for effective vaccine administration, and functional cold chain network to maintain vaccine viability. These problems have led to heightened interest in producing new antiviral strategies, such as RNA interference (RNAi). RNAi methodology is capable of substantially decreasing viral replication at a cellular level, both in vitro and in vivo. In this study, we utilize microRNA (miRNA)-expressing constructs (a type of RNA interference) in an attempt to target and knockdown five NDV structural RNAs for nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), and large (L) protein genes. Immortalized chicken embryo fibroblast cells (DF-1) that transiently expressed miRNA targeting NP mRNA, showed increased resistance to NDV-induced cytopathic effects, as determined by cell count, relative to the same cells expressing miRNA against alternative NDV proteins. Upon infection with NDV, DF-1 cells constitutively expressing the NP miRNA construct had improved cell survival up to 48 h post infection (h.p.i) and decreased viral yield up to 24 h.p.i. These results suggest that overexpression of the NP miRNA in cells and perhaps live animal may provide resistance to NDV.

Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines.

While there is typically 100% survivability in birds challenged with vNDV under experimental conditions, either with vaccines formulated with a strain homologous or heterologous (different genotype) to the challenge virus, vaccine deficiencies are often noted in the field. We have developed an improved and more stringent protocol to experimentally evaluate live NDV vaccines, and showed for the first time under experimental conditions that a statistically significant reduction in mortality can be detected with genotype matched vaccines. Using both vaccine evaluation protocols (traditional and improved), birds were challenged with a vNDV of genotype XIII and the efficacy of live heterologous (genotype II) and homologous (genotype XIII) NDV vaccines was compared. Under traditional vaccination conditions there were no differences in survival upon challenge, but the homologous vaccine induced significantly higher levels of antibodies specific to the challenge virus. With the more stringent challenge system (multiple vaccine doses and early challenge with high titers of vNDV), the birds administered the homologous vaccine had superior humoral responses, reduced clinical signs, and reduced mortality levels than those vaccinated with the heterologous vaccine. These results provide basis for the implementation of more sensitive methods to evaluate vaccine efficacy.

Separate evolution of virulent newcastle disease viruses from Mexico and Central America.

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.