Intraocular Route Research Articles

A comparative evaluation of serum biochemistry profile and antigenic relatedness among velogenic and mesogenic Avian avulavirus 1 infection in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10-6.51 EID50/0.1mL for sub-genotype VIIi and 10-6.87 EID50/0.1mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (p< 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (p< 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (p< 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (R-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.

A Novel Adjuvant "Sublancin" Enhances Immune Response in Specific Pathogen-Free Broiler Chickens Inoculated with Newcastle Disease Vaccine.

Sublancin is a glycosylated antimicrobial peptide produced by Bacillus subtilis 168 possessing antibacterial and immunomodulatory activities. This study was aimed at investigating the effects of sublancin on immune functions and serum antibody titer in specific pathogen-free (SPF) broiler chickens vaccinated with Newcastle disease (ND) vaccine. For this purpose, 3 experiments were performed. Experiment 1: SPF broiler chicks (14 days old) were randomly allotted to 1 of 7 groups including a blank control (BC), vaccine control (VC), and 5 (3-7) vaccinated and sublancin supplemented at 5, 15, 30, 45, and 60 mg activity/L of water, respectively. Vaccinated groups (2-7) were vaccinated with ND vaccine by intranasal and intraocular routes at the 14th day. On 7, 14, 21, and 28 days post vaccination (dpv), the blood samples were collected for the determination of serum hemagglutination inhibition (HI) antibody titer. Experiment 2: SPF broiler chicks were divided into 1 of 3 groups, i.e., blank control (BC), vaccine control (VC), and sublancin treatment (ST). On 7, 14, and 21 dpv, the blood samples were collected for measuring HI antibody titer by micromethod. Experiment 3: the design of this experiment was the same as that of experiment 2. On 7 and 21 dpv, pinocytosis of peritoneal macrophages, B lymphocyte proliferation assay, measurement of CD4+ and CD8+ T cells, and serum cytokine quantitation were carried out. It was noted that sublancin promoted B lymphocyte proliferation, increased the proportion of CD8+ T lymphocyte subpopulations, and enhanced the antibody titer in broiler chickens. In addition, it was also observed that sublancin has the potential to induce the secretion of IFN-γ, IL-10, and IL-4. In conclusion, these findings suggested that sublancin could promote both humoral and cellular immune responses and has the potential to be a promising vaccine adjuvant.

Effect of acute and chronic aldosterone exposure on the retinal pigment epithelium-choroid complex in rodents

Preclinical and clinical evidences show that aldosterone and/or mineralocorticoid receptor (MR) over-activation by glucocorticoids can be deleterious to the retina and to the retinal pigment epithelium (RPE)-choroid complex. However, the exact molecular mechanisms driving these effects remain poorly understood and pathological consequences of chronic exposure of the retina and RPE/choroid to aldosterone have not been completely explored. We aimed to decipher the transcriptomic regulation in the RPE-choroid complex in rats in response to acute intraocular aldosterone injection and to explore the consequences of systemic chronic aldosterone exposure on the morphology and the gene regulation in RPE/choroid in mice. High dose of aldosterone (100 nM) was intravitreously injected in Lewis rat eyes in order to yield an aldosterone dose able to induce a molecular response at the apical side of the RPE-choroid complex. The posterior segment morphology was evaluated in vivo using optical coherence tomography (OCT) before and 24 h after aldosterone injection. Rat RPE-choroid complexes were used for RNA sequencing and analysis. Uninephrectomy/aldosterone/salt (NAS) model was created in wild-type C57BL/6 mice. After 6 weeks, histology of mouse posterior segments were observed ex vivo. Gene expression in the RPE-choroid complex was analyzed using quantitative PCR. Acute intravitreous injection of aldosterone induced posterior segment inflammation observed on OCT. RNA sequencing of rat RPE-choroid complexes revealed up-regulation of pathways involved in inflammation, oxidative stress and RNA procession, and down-regulation of genes involved in synaptic activity, muscle contraction, cytoskeleton, cell junction and transporters. Chronic aldosterone/salt exposure in NAS model induces retinal edema, choroidal vasodilation and RPE cell dysfunction and migration. Quantitative PCR showed deregulation of genes involved in inflammatory response, oxidative stress, particularly the NOX pathway, angiogenesis and cell contractility. Both rodent models share some common phenotypes and molecular regulations in the RPE-choroid complex that could contribute to pachychoroid epitheliopathy in humans. The difference in inflammatory status relies on different intraocular or systemic route of aldosterone administration and on the different doses of aldosterone exposed to the RPE-choroid complex.

Has retinal gene therapy come of age? From bench to bedside and back to bench.

Retinal gene therapy has advanced considerably in the past three decades. Initial efforts have been devoted to comprehensively explore and optimize the transduction abilities of gene delivery vectors, define the appropriate intraocular administration routes and obtain evidence of efficacy in animal models of inherited retinal diseases (IRDs). Successful translation in clinical trials of the initial promising proof-of-concept studies led to the important milestone of the first approved product for retinal gene therapy in both US and Europe. The unprecedented clinical development observed during the last decade in the field is however highlighting new challenges that will need to be overcome to bring gene therapy to fruition to a larger patient population within and beyond the realm of IRDs.

Conservative management of retinoblastoma: Challenging orthodoxy without compromising the state of metastatic grace. “Alive, with good vision and no comorbidity”

Retinoblastoma is lethal by metastasis if left untreated, so the primary goal of therapy is to preserve life, with ocular survival, visual preservation and quality of life as secondary aims. Historically, enucleation was the first successful therapeutic approach to decrease mortality, followed over 100 years ago by the first eye salvage attempts with radiotherapy. This led to the empiric delineation of a window for conservative management subject to a "state of metastatic grace" never to be violated. Over the last two decades, conservative management of retinoblastoma witnessed an impressive acceleration of improvements, culminating in two major paradigm shifts in therapeutic strategy. Firstly, the introduction of systemic chemotherapy and focal treatments in the late 1990s enabled radiotherapy to be progressively abandoned. Around 10 years later, the advent of chemotherapy in situ, with the capitalization of new routes of targeted drug delivery, namely intra-arterial, intravitreal and now intracameral injections, allowed significant increase in eye preservation rate, definitive eradication of radiotherapy and reduction of systemic chemotherapy. Here we intend to review the relevant knowledge susceptible to improve the conservative management of retinoblastoma in compliance with the "state of metastatic grace", with particular attention to (i) reviewing how new imaging modalities impact the frontiers of conservative management, (ii) dissecting retinoblastoma genesis, growth patterns, and intraocular routes of tumor propagation, (iii) assessing major therapeutic changes and trends, (iv) proposing a classification of relapsing retinoblastoma, (v) examining treatable/preventable disease-related or treatment-induced complications, and (vi) appraising new therapeutic targets and concepts, as well as liquid biopsy potentiality.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. Results in mice showed >300days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Variations of immunoglobulin A in serum, tracheal, and intestinal tissues in chickens following inoculation of two strains of Newcastle disease vaccines

To elucidate secretory immunoglobulin A (IgA) response as a part of mucosal immunity, it is decided to compare the effectiveness of intraocular administration of Hitchner B1 and thermostable I-2 vaccines in broiler chickens challenged with viscerotropic velogenic Newcastle disease virus (vvNDV) (Herts33). For this purpose, 301-day-old chicks were randomly divided in to the four groups. Groups 1 and 2 were inoculated with I-2 and HB1 vaccines twice via intraocular route with 1-week intervals, and groups 3 and 4 were considered positive and negative controls and birds were challenged 2 weeks after the second inoculation. Protection rate in I-2 and HB1 vaccine groups was 100% and 97% respectively with no significant differences (P > 0.05). All vaccinated groups showed elevation in ND HI antibody titer after vaccination. There were no significant differences (P > 0.05) in ND HI antibody between two vaccinated groups. First levels of detectable total IgA in a serum sample were observed 3 dpv1. No significant differences (P > 0.05) were observed between two vaccinated groups in IgA antibody titer in sera. The vaccinated groups were evaluated by IHC for detection IgA in the trachea and intestine. There were some differences between the vaccinated groups but these differences were not significant. Experimental studies have shown that both vaccines induce antibody production in mucosal surface and circulatory system and thermostable I-2 vaccine could be successfully used in the broiler chicken as effective as HB1 vaccine, so I-2 vaccine could be a suitable heat resistance candidate to reduce mortality in the poultry industry faced to vvND.

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers.

Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan-adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham-negative and sham-positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1mL containing Thai MG strain 107 colony-forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real-time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin.

Acute phase proteins and stress markers in the immediate response to a combined vaccination against Newcastle disease and infectious bronchitis viruses in specific pathogen free (SPF) layer chicks

Vaccination is an important tool in poultry health, but is itself a stressor often resulting in a reduction in feed intake, body weight gain, and nutrient digestibility. In other species, vaccination is associated with an immediate acute-phase response. As an important immune parameter, the circulating heterophil/lymphocyte (H/L) ratio is a well-recognized parameter of stress in poultry. In this study, the effects of a routinely used commercial poultry vaccine on the acute phase response (APR) and H/L ratios in specific pathogen-free (SPF) layer chicks was examined to determine if post vaccination (PV) stress and an APR occur. A combined Newcastle disease and infectious bronchitis vaccine (Nobalis Ma5+Clone 30) was administered to SPF chicks by the intraocular route at age 7 d. Acute phase proteins (APP), alpha-1 acid glycoprotein (AGP) and serum amyloid A (SAA) were measured by enzyme-linked immunosorbent assays at d 0 (pre-vaccination) and d 0.5, 1, 2, 3, 4, 5, 6, and 21 PV. Stress was determined in the chicks by measurement of the H/L ratio. The immune response to the vaccine was estimated by measurement of the antibody (IgY) response to the vaccine at d 21.The antibody titer was significantly (P < 0.05) higher in the vaccinated group at 21 d PV, confirming stimulation of the immune system. The H/L ratio was also significantly higher in the vaccinated group at 1 to 2 d (P < 0.01) and at 3 d (P < 0.05) PV. The concentration of SAA increased by 2.8-fold, from 63.7 μg/mL in controls to 181 μg/mL in the vaccinated group, (P < 0.05) at 1 d PV. AGP increased 1.6-fold at 2 d PV, (from 0.75 g/mL in the control group to 1.24 g/mL in the vaccinated group, P < 0.05).In conclusion an immediate but mild APR occurred in the chicks following intraocular vaccination, whereas the stress response as measured by H/L ratio seemed to be more specific and sensitive. Measurement of these biomarkers of the host response could be a tool in vaccine development.

Adipose mesenchymal stem cells-derived exosomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits.

This study aimed to evaluate the effect of mesenchymal stem cells (MSCs)–derived exosomes in retina regeneration of experimentally induced diabetes mellitus (DM) in a rabbit model. Exosomes are extracellular vesicles that contain many microRNAs (micRNAs), mRNAs, and proteins from their cells of origin. DM was induced by intravenous (IV) injection of streptozotocin in rabbits. MSCs were isolated from adipose tissue of rabbits. Exosomes were extracted from MSCs by ultracentrifugation. Exosomes were injected by different routes (IV, subconjunctival (SC), and intraocular (IO)). Evaluation of the treatment was carried out by histopathological examination of retinal tissues and assessment of micRNA-222 expression level in retinal tissue by real-time polymerase chain reaction. Histologically, by 12 weeks following SC exosomal treatment, the cellular components of the retina were organized in well-defined layers, while IO exosomal injection showed well-defined retinal layers which were obviously similar to layers of the normal retina. However, the retina appeared after IV exosomal injection as irregular ganglionic layer with increased thickness. MicRNA-222 expression level was significantly reduced in diabetic controls when compared to each of healthy controls and other diabetic groups with IV, SC, and IO routes of injected exosomes (0.06 ± 0.02 vs. 0.51 ± 0.07, 0.28 ± 0.08, 0.48 ± 0.06, and 0.42 ± 0.11, respectively). We detected a significant negative correlation between serum glucose and retinal tissue micRNA-222 expression level (r = −0.749, p = 0.001). We can associate the increased expression of micRNA-222 with regenerative changes of retina following administration of MSCs-derived exosomes. The study demonstrates the potency of rabbit adipose tissue–derived MSCs exosomes in retinal repair. So, exosomes are considered as novel therapeutic vectors in MSCs-based therapy through its role in shuttling of many factors including micRNA-222.

The positive expression of genotype VII Newcastle disease virus (Malaysian isolate) in Japanese quails (Coturnix coturnix japonica).

Aim:Genotype VII Newcastle disease virus (NDV) is the most predominant NDV strains that circulating in Malaysia; thus, this study was aimed to determine the susceptibility of Japanese quails toward genotype VII NDV. Clinical signs, gross pathological lesions of organs, positive detection of virus in organs and cloacal swabs, as well as the expression of the antibody titer, were used as parameters to assess the susceptibility of Japanese quails following infection of genotype VII NDV.Materials and Methods:About 20 quails were divided into three groups (n=8 for Groups A and B; n=4 for the control group). The quails in the Groups A and B were infected via intraocular route with 0.03 ml of 103.5 ELD50 and 107.0 ELD50 of NDV strain IBS 002, respectively, while the control group received 1× phosphate-buffered saline. Cloacal swabs and necropsy were taken on day 7 post-infection for all quails were subjected to one-step reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) for detection of virus and examination for gross pathological lesion, respectively. Blood serums of infected quails were taken on day 10, 14, and 21 post-day infections and were subjected for hemagglutination inhibition (HI) assay.Results:Depression and ruffled feathers, trachea rales, leg paralysis, and torticollis were shown in some of the quails in both infected groups. Based on statistical analysis, there was no significant difference (p>0.05) in clinical signs between the infected groups. The results for RT-qPCR were found to be negative for all groups, and no gross pathological lesions of organs observed for quails in both infected groups. Trachea, proventriculus, and cecal tonsil were taken for the detection of NDV by RT-qPCR, and some of the organ samples showed positive detection of virus in both infected groups. HI assay showed an increase in mean titers of antibody across time and between infected groups.Conclusion:In summary, Japanese quails are susceptible to genotype VII NDV based on parameters assessed.

Deterioration of eggshell quality is related to calbindin in laying hens infected with velogenic genotype VIId Newcastle disease virus

The aim of this study was to determine the mechanism by which Newcastle disease virus (NDV) affects eggshell quality. Thirty-week-old specific pathogen free (SPF) egg-laying hens were inoculated with the velogenic genotype VIId NDV strain (infected group) or with inoculating media without virus (control group) by combined intraocular and intranasal routes. The levels of CaBP-D28k mRNA expression in the uterus, a gene related to eggshell quality, were examined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The quality of eggshells was analyzed by scanning electron microscopy (SEM). The infected group showed a marked decline in egg production when compared to the control group. The NDV antigen was found more abundantly in the glandular epithelium of the infected hens' uteri from 1 to 15 d post-inoculation (dpi). The levels of CaBP-D28k mRNA expression in the uteri of infected hens were significantly lower than in the control hens from 3 to 15 dpi (P < 0.05). The changes in the Ca concentrations in the eggshells were consistent with the expression of CaBP-D28k mRNA in the infected hens. Ultrastructural examination of eggshells showed significantly reduced shell thickness in the infected hens from 1 to 15 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens compared with the control hens. In conclusion, the current study confirmed that velogenic genotype VIId NDV infection is associated with the deterioration of the eggshell quality of the laying hens.

Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Takotsubo Cardiomyopathy Due to Systemic Absorption of Intraocular Phenylephrine

We present a case of Takotsubo cardiomyopathy (TTC) with regional variation in left ventricular function precipitated by intraocular injection of phenylephrine. To our knowledge, this is the first described case of TTC occurring in the setting of an intraocular medication. Contrary to the traditional model of the β2-receptor underlying the pathophysiology behind Takotsubo cardiomyopathy, phenylephrine is a pure α1-agonist implicating alternative mechanisms of myocardial injury resulting in a similar clinical phenotype. One should be alert to the possibility of catecholamine induced TTC occurring in patients subject to medications administered via the intraocular route with the potential for significant systemic absorption.

Deterioration of eggshell quality in laying hens experimentally infected with H9N2 avian influenza virus.

This study aimed to determine the mechanism by which H9N2 avian influenza virus (AIV) affects eggshell quality. Thirty-week-old specific pathogen free egg-laying hens were inoculated with the chicken-origin H9N2 AIV strain (A/Chicken/shaanxi/01/2011) or with inoculating media without virus by combined intraocular and intranasal routes. The time course for the appearance of viral antigen and tissue lesions in the oviduct was coincident with the adverse changes in egg production in the infected hens. The viral loads of AIV have a close correlation with the changes in the uterus CaBP-D28k mRNA expression as well as the Ca concentrations in the eggshells in the infected hens from 1 to 7 days post inoculation (dpi). Ultrastructural examination of eggshells showed significantly decreased shell thickness in the infected hens from 1 to 5 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens from 1 to 5 dpi as compared with the control hens. In conclusion, this study confirmed that H9N2 AIV strain (A/Chicken/shaanxi/01/2011) infection is associated with severe lesions of the uterus and abnormal expression of CaBP-D28k mRNA in the uteri of the infected hens. The change of CaBP-D28k mRNA expression may contribute to the deterioration of the eggshell quality of the laying hens infected with AIV. It is noteworthy that the pathogenicity of H9N2 AIV strains may vary depending on the virus strain and host preference.

Phylogenetic and Pathotypic Characterization of Newcastle Disease Viruses Circulating in South China and Transmission in Different Birds.

Although Newcastle disease virus (NDV) with high pathogenicity has frequently been isolated in poultry in China since 1948, the mode of its transmission among avian species remains largely unknown. Given that various wild bird species have been implicated as sources of transmission, in this study we genotypically and pathotypically characterized 23 NDV isolates collected from chickens, ducks, and pigeons in live bird markets (LBMs) in South China as part of an H7N9 surveillance program during December 2013–February 2014. To simulate the natural transmission of different kinds of animals in LBMs, we selected three representative NDVs—namely, GM, YF18, and GZ289—isolated from different birds to evaluate the pathogenicity and transmission of the indicated viruses in chickens, ducks, and pigeons. Furthermore, to investigate the replication and shedding of NDV in poultry, we inoculated the chickens, ducks, and pigeons with 106 EID50 of each virus via intraocular and intranasal routes. Eight hour after infection, the naïve contact groups were housed with those inoculated with each of the viruses as a means to monitor contact transmission. Our results indicated that genetically diverse viruses circulate in LBMs in South China's Guangdong Province and that NDV from different birds have different tissue tropisms and host ranges when transmitted in different birds. We therefore propose the continuous epidemiological surveillance of LBMs to support the prevention of the spread of these viruses in different birds, especially chickens, and highlight the need for studies of the virus–host relationship.

Allogeneic Mesenchymal Stem Cell Treatment Induces Specific Alloantibodies in Horses.

Background. It is unknown whether horses that receive allogeneic mesenchymal stem cells (MSCs) injections develop specific humoral immune response. Our goal was to develop and validate a flow cytometric MSC crossmatch procedure and to determine if horses that received allogeneic MSCs in a clinical setting developed measurable antibodies following MSC administration. Methods. Serum was collected from a total of 19 horses enrolled in 3 different research projects. Horses in the 3 studies all received unmatched allogeneic MSCs. Bone marrow (BM) or adipose tissue derived MSCs (ad-MSCs) were administered via intravenous, intra-arterial, intratendon, or intraocular routes. Anti-MSCs and anti-bovine serum albumin antibodies were detected via flow cytometry and ELISA, respectively. Results. Overall, anti-MSC antibodies were detected in 37% of the horses. The majority of horses (89%) were positive for anti-bovine serum albumin (BSA) antibodies prior to and after MSC injection. Finally, there was no correlation between the amount of anti-BSA antibody and the development of anti-MSC antibodies. Conclusion. Anti allo-MSC antibody development was common; however, the significance of these antibodies is unknown. There was no correlation between either the presence or absence of antibodies and the percent antibody binding to MSCs and any adverse reaction to a MSC injection.

Apoptosis induction and release of inflammatory cytokines in the oviduct of egg-laying hens experimentally infected with H9N2 avian influenza virus

The H9N2 subtype avian influenza virus (AIV) can cause serious damage to the reproductive tract of egg-laying hens, leading to severe egg-drop and poor egg shell quality. However, previous studies in relation to the oviductal-dysfunction resulted from this agent have not clearly been elucidated. In this study, apoptosis and pathologic changes in the oviducts of egg-laying hens caused by H9N2 AIV were evaluated. To understand the immune response in the pathogenic processes, 30-week old specific pathogen free (SPF) egg-laying hens inoculated with H9N2 subtype of AIV through combined intra-ocular and intra-nasal routes. H9N2 AIV infection resulted in oviductal lesions, triggered apoptosis and expression of immune related genes accompanied with infiltration of CD3+CD4+ and CD3+CD8α+ cells. Significant tissue damage and apoptosis were observed in the five oviductal parts (infundibulum, magnum, isthmus, uterus and vagina) at 5 days post-inoculation (dpi). Furthermore, immune-related genes, including chicken TLR3 (7, 21), MDA5, IL-2, IFN-β, CXCLi1, CXCLi2, XCL1, XCR1 and CCR5 showed variation in the egg-laying hens infected with H9N2 AIV. Notably, mRNA expression of IFN-α was suppressed during the infection. These results show distinct expression patterns of inflammatory cytokines and chemokines amongst segments of the oviduct. Differential gene expression of inflammatory cytokines and lymphocytes aggregation occurring in oviducts may initiate the infected tissue in response to virus replication which may eventually lead to excessive cellular apoptosis and tissue damage.

Effect of vaccination on transmission characteristics of highly virulent Newcastle disease virus in experimentally infected chickens

An experimental study was conducted to evaluate the effect of vaccines produced in Ethiopia from vaccine strains used worldwide on the transmission characteristics of velogenic Newcastle disease virus field strain after different vaccination schemes. Chickens were vaccinated with Hitchner B1, La Sota or I-2 via the intraocular and intranasal routes. Vaccine and challenge viruses induced high antibody levels, both in inoculated and contact birds. Prime-boost vaccination protected birds against morbidity and mortality and significantly reduced the incidence of viral shedding from chickens compared with single vaccinated and unvaccinated birds. Protection from disease and mortality was correlated with the presence of positive antibody titres (>4 log2) at day of challenge. Most of the unvaccinated and in-contact birds excreted the virus and showed a high level of antibody titres, indicating the high infectivity of the challenge virus. The detection of the challenge virus in most of vaccinated birds demonstrated that the tested vaccination protocols cannot fully protect birds from viral infection, replication and shedding, and vaccinated–infected birds can act as a source of infection for susceptible flocks. The high mortality observed in unvaccinated birds and their contacts confirmed the virulence of the challenge virus and indicated that this field virus strain can easily spread in an unvaccinated poultry population and cause major outbreaks. Progressive vaccinations supported by biosecurity measures should therefore be implemented to control the disease and introduction of the virus to the poultry farms.