Intraocular Route Research Articles

Development of a low-dose fast-dissolving tablet formulation of Newcastle disease vaccine for low-cost backyard poultry immunisation

The immunisation of backyard poultry is critical for maintaining healthy flocks to provide nutrition and income for low-resource farmers worldwide. A vaccine presentation for flocks of less than 50 birds...

The in vivo Effect of Oselatmivir against Highly Pathogenic Avian Influenza Virus (H5N1) Isolated from Egypt.

Highly pathogenic avian influenza is still a major problem in poultry sector and house hold chickens in Egypt. This study was directed to detect and characterize the AIVs circulating among chickens flocks in our region (Al-Sharkia Governorate) during 2006-2011. Samples were collected from 31 poultry flock, based on the immune-chromatographic assay, virus isolation and HI test samples from 17 chickens flocks were positive (54.8%) and characterized as AIV type A subtype H5. For further characterization all samples were investigated using RT-PCR, the results revealed that all isolated viruses were of H5N1 AIV subtype. To investigate the in vivo effect of Oselatmivir® against HPAI(H5N1) in chickens, 120 birds were divided into 6 groups (n=6) 20 birds/group. Birds of groups I,II and III were vaccinated while birds of groups IV,V and VI were kept unvaccinated. Birds were challenged using AIA/Chicken/Egypt/ 101/H5N1/2011via intra-ocular route with a dose of 105/0.1 ml ELD50 per bird. Oselatmivir treatment (5mg oseltamivir phosphate/bird) were given to birds of groups I,II,IV and VI at different duration while, birds of groups III and VI received PBS at the same schedule. Results revealed that oseltamivir was able to reduce the virus replication, clinical disease and virus shedding especially in vaccinated bird.

Evaluation of the shedding routes and serological patterns in experimentally-induced Brucella melitensis infection in dexamethasone-treated and transport-stressed goats

Aim: To identify and evaluate the shedding routes and patterns following experimentally-induced Brucella melitensis infection in dexamethasone-treated and transport-stressed goats. Materials and Methods: Twenty four healthy, adult goats were divided into 4 groups: A, B, C and D respectively. Group A 7 was treated with dexamethasone for 8 days prior to inoculation with 10 Colony Forming Units of B. melitensis via the intraocular route. Group B was transported for 3 hours prior to inoculation with a similar dose. Group C was inoculated with a similar dose without subjecting the animals to any prior treatment, and this group served as our positive control. Group D was not inoculated with the infective dose and served as our negative control. Blood samples along with nasal, ocular, and vaginal swabs were collected on days 0, 3, 7, 10, 14, and weekly thereafter until day 63 post inoculation (pi) and were analyzed by PCR, Rose Bengal Plate Test (RBPT), and indirect ELISA techniques. Results: The nasal, ocular and vaginal swabs tested positive for Brucellosis with PCR from day 7, with nasal route being the first and most consistent route to reveal the positive results. Group B showed the earliest onset of shedding the bacterium (day 7) followed by group A which started from day 10 and shed relatively more positive of the bacterium via the routes examined. Blood samples tested positive with PCR from day 7 through 14 and the results were inconsistent subsequently. Sera samples tested positive with RBPT on day 14 in all the 3 infected groups but more consistent in group C. On the other hand, tests using ELISA showed positive results from day 7 pi, with group C having a 100% seroconvertion –while groups A and B showed only 50% seroconvertion. Conclusion: The consistent shedding via the nasal, ocular, and vaginal routes in groups A and B implied possible immunosuppression in the infected animals. We recommend that programs designed to control Brucellosis should consider analyzing a larger number of biological samples to enhance the accuracy of identification of shedders.

Immune response and protective potential following vaccination against Newcastle disease virus and fowl cholera in Naked neck chickens

Humoral immune responses to Newcastle Disease Virus (NDV) and Bangladesh Agricultural University (BAU) Fowl cholera (BAUFC) vaccines were evaluated in naked neck chickens (NNC). Ten birds were vaccinated with Baby Chick Ranikhet Disease Virus (BCRDV) at Day 7 through intra-ocular route and with Ranikhet Disease Virus (RDV) at day 35 of age through intramuscular route. Serum antibodies were measured by Haemagglutination Inhibition test. Two weeks after final immunization all birds were challenged with virulent field isolate of NDV where all vaccinated birds survived without illness during ten days, and all ten control birds died. Ten birds were vaccinated with BAUFC vaccine at Day 42 and 70 according to the Manufacturer’s instruction, which induced detectable levels of antibody titre as determined by Passive Haemagglutination Assay (PHA) test. Eight vaccinated birds survived following challenge with virulent fowl cholera isolate two weeks after final vaccination and all ten control birds died. DOI: http://dx.doi.org/10.3329/bvet.v29i2.14342 Bangl. vet. 2012. Vol. 29, No. 2, 49-55

The Efficacy of Ranikhet Disease Vaccines Produced by Livestock Research Institute of Bangladesh

The study was conducted to investigate the efficacy of Baby chick Ranikhet Disease Vaccine (BCRDV) and Ranikhet Disease Vaccine (RDV) produced by the Livestock Research Institute (LRI), Mohakhali, Dhaka. For this experiment, 100 day-old-chick was purchased from Phinex Hatchery Ltd., Gazipur. The chicks (n=100) were divided into two groups. In group A (n=50), vaccination was performed twice with BCRDV at 2 and 21 days of age through intraocular route (i/o) followed by once with RDV at 60-day of age through intramuscular (i/m) route. Group B (n=50) was kept as unvaccinated control. The immunogenicity of the vaccine was evaluated by measuring the serum HI antibody titers at 1-, 20-, 36-, and 76-day of age, while the vaccine efficacy was examined by a challenge infection experiment with a velogenic field isolate of NDV as well as passive protection test. It was observed that the maternal antibody titers of the unvaccinated control group B gradually declined from day 1 to day 76 of age. Conversely, after primary and secondary vaccination with BCRDV, the levels of serum HI titer slightly increased in vaccinated group A compared with those in control group B. Finally administration of RDV resulted in a sharp increase in HI titer, leading to protection from challenge infection with virulent field virus as well as passive protection test. These results clearly demonstrated that a prime-booster immunization with BCRDV and RDV, both produced by LRI, is effective to protect chicken against Newcastle disease (ND).DOI: http://dx.doi.org/10.3329/mh.v1i1.13706 Microbes and Health Vol.1(1) June 2012 pp.9-13

Comparative Studies on the Oral and Intraocular Routes of Administration of Newcastle Disease Vaccine, La Sota in Adult Chickens.

Two hundred and fifty (250) white cockerels were used to compare the differences between the intraocular and oral routes of administration of ND vaccine La Sota in adult chickens. At 20 weeks of age, serum samples were collected from the chickens and used to estimate the antibody levels against Newcastle disease virus using heamagglutination inhibition (HI) test. The birds were randomly divided into two groups, G1 and G2. Birds in G1 were vaccinated with ND vaccine La Sota intraocularly while birds in G2 were vaccinated orally. Serum samples were collected from chickens in the two groups weekly and used to estimate the HI antibody. Result showed higher antibody level in the chickens vaccinated intraocularly when compared with those vaccinated orally. There was significant difference (p < 0.05) between the geometric HI titres of chickens in the two groups on days 7, 14 and 28 PV but it did not differ significantly on day 21 PV. Challenge studies showed 100% protection of chicken in the two groups on day 21 PV. We identified Harderian glands' possible roles in producing local and humoral immunity in intraocular vaccinations and the destruction of some of the vaccinal viruses by gastric secretions in oral vaccinations as the possible reasons for the production of higher antibodies in intraocular vaccination in adult chickens.

Intravitreal clindamycin plus dexamethasone versus classic oral therapy in toxoplasmic retinochoroiditis: a prospective randomized clinical trial

Both oral and intraocular routes have been recommended for medication administration in toxoplasmic retinochoroiditis; however, available data, in support or against, are scarce. The objective of this study was to compare the efficacy of intravitreal clindamycin plus dexamethasone (IVCD) and conventional oral therapy (COT) including pyrimethamine, sulfadiazine, folinic acid and prednisone in active toxoplasmic retinochoroiditis. In this prospective randomized single-blind clinical trial, patients with active toxoplasmic retinochoroiditis received either IVCD (n=32), or COT (n=34) for 6weeks. Changes of best-corrected visual acuity, retinal lesion size, and vitreous inflammation before and after treatment, as well as complications/side-effects and recurrence rate within at least 2years of follow-up were compared between groups. Although all the variables improved significantly at 6weeks within each group, changes were comparable between the IVCD and COT receivers. There was only one case with hepatotoxicity in the COT group which responded favorably to drug change. No injection-related complication was observed. Recurrence rates were 12.5 and 14.7% in the IVCD and COT groups, respectively (p=0.54). In conclusion, both IVCD and COT are equally effective against active toxoplasmic retinochoroiditis but the former is apparently safer and more convenient.

Intraocular administration of biodegradable microspheres to increase drug bioavailability and extend therapeutic effect

AbstractPurpose Treatment of posterior segment diseases often requires repeated intravitreal injections to achieve and maintain steady state concentrations of the active substance in the target site. However, intraocular injections are associated to adverse effects and the risk of complications increases with the number of administrations. Biodegradable microspheres (MPs) are considered an alternative to multiple injections as they are able to release the encapsulated drug over weeks or months. Copolymers of lactic and glycolic acids (PLGA) are FDA approved biodegradable biomaterials commonly used to prepare MPs. They are biocompatible and degrade to metabolic products that are easily eliminated from the body. The main advantages of these systems is that they can be injected as a conventional suspension without surgical procedures. After intravitreal administration, PLGA microspheres suffer aggregation disappearing from the site of injection after delivering the drug. Microspheres can be loaded with different active substances (small molecules and biotechnological products) useful to treat vitreoretinal diseases. Administration of microparticles can be performed by periocular, intravitreal, sub‐ retinal, or other intraocular routes to treat posterior segment disorders.Conclusion PLGA microspheres are emerging therapeutic tools for the treatment of posterior segment diseases in which steady state concentrations of the active substance is needed over an extended period of time. Acknowledgements: PANOPTES‐Peptide‐based Nanoparticles as Ocular Drug Delivery Vehicles‐ collaborative project (FP7‐NMP‐2009); Spanish Ministry of Economy MAT 2010‐18242, UCM Research Group 920415 and RETICs (RD07/0062/2002)

IMMUNOGENIC RESPONSE WITH EFFICACY OF CERTAIN GUMBORO VACCINES IN BROILER

The research work was carried out to determine the immunogenic response with efficacy of six commercial Gumboro, vaccines (Nobilis® Gumboro D78 and Nobilis® Gumboro 228E, Intervet, The Netherlands; Bur-706®, Merial, France; TAD Gumboro vac® and TAD Gumboro vac forte®, Lohmann Animal Health, Germany; BursaplexTM, Merial Select, Inc. Gainesville, USA) under experimental condition in broiler chickens during the period from November 2002 to May 2003. The chickens of two groups were vaccinated with Nobilis® Gumboro D78 and Nobilis® Gumboro 228E at 14 days of age with a booster dose at 21 days of age and were challenged at 40 days of age with vvIBDV. TAD Gumboro vac® and TAD Gumboro vac forte® were inoculated into the two groups of chickens at 14 and 28 days of age and the chickens were challenged at 35 days of age with vvIBDV. Bur-706® vaccine was given to the chickens of another group at one-day-old and 14 days of age and the chickens were challenged at 28 days of age. One group of chickens was vaccinated with Bursaplex™ vaccine at 1-day-old with no booster dose and was challenged at 21 days of age. The percentage of protection in birds receiving Nobilis® Gumboro D78 and Nobilis® Gumboro 228E vaccines by intraocular route was 92.30 and 96.29 respectively, whereas the percentages of protection in birds receiving TAD Gumboro vac®, TAD Gumboro, vac forte® and Bur-706® vaccines were 95.65, 100 and 95.83 respectively. BursaplxTM provided 100% protection against challenge with vvIBDV. Chickens vaccinated with Nobilis® Gumboro D78, Nobilis® Gumboro 228E, TAD Gumboro vac®, and Bur-706®, showed significant (p &lt; 0.05, p &lt; 0.01) decrease in ELISA antibody titre up to the day of challenge infection. BursaplexTM vaccinated group also showed significant decrease in ELISA antibody titre by 7 days (p &lt; 0.05) and by 14 and 21 days (p &lt; 0.01) after primary vaccination. In case of TAD Gumboro vac forte®, 7 days (839 ± 219.34) and 14 days (258 ± 44.80) after primary vaccination, the ELISA antibody titre significantly (p &lt; 0.01) decreased but 7 days after booster vaccination, the ELISA antibody titre significantly (p &lt; 0.01) increased (1299 ± 37.51). All the control sera revealed significant (p &lt; 0.01) decrease of ELISA antibody titre up to day of challenge infection. There was an insignificant increase in ELISA antibody titre 7 days after booster vaccination in case of unvaccinated control groups for TAD Gumboro vac® and TAD Gumboro vac forte® only. Nobilis® Gumboro D78, TAD Gumboro vac® and Bur-706® vaccinated groups revealed significant (p &lt; 0.01) increase of ELISA antibody titre 7 days post-challenge while Nobilis® Gumboro 228E and BursaplexTM vaccinated groups showed significant (p &lt; 0.05) increase of antibody titre after 7 days of challenge. Insignificant increase of antibody titre was recorded in TAD Gumboro vac forte® vaccinated group. It may be concluded that TAD Gumboro vac forte® (intermediate plus) and BursaplexTM (Merial Select, Inc. Gainesville, USA) can be used to immunize the broiler birds sufficiently against IBD. However, it is necessary to estimate the optimum vaccination timing, i.e., to determine when maternal antibodies in chicks will decline to levels that the vaccines can overcome.

EFFECT OF VITAMIN-MINERAL PREMIX ON IMMUNE RESPONSE OF BABY CHICK RANIKHET DISEASE VACCINE IN CHICKS

A total of 150 (75 broiler and 75 layer) apparently healthy chicks were used to investigate the effect of vitamin-mineral premix on immune responses of Newcastle disease (ND) vaccine (Baby Chick Ranikhet Disease Vaccine, F-strain) during the period from January to March 2003. The chicks were divided into six groups namely group A, B and C for broiler and group D, E and F for layer birds consisting of 25 birds in each group. Chicks of group A, C, D and F were supplied with vitamin-mineral premix containing feed and chicks of group B and E were supplied with vitamin-mineral deficient feed. The chicks of group A, B, D and E were primarily vaccinated with 0. 1 ml of BCRDV of 1065 EID50/0. 1 ml per bird through intraocular route at the age of day 3 and were revaccinated with the same vaccine at the age of day 17. Birds of group C and F were kept as unvaccinated control. Chickens of all the groups were challenged with the virulent isolate of NDV @ 0.5 ml/bird IM (1045 EID50/0.1 ml) after two weeks of secondary vaccination. Titres were determined before and after vaccination by hemagglutination test (HI) test and serum neutralization test (SNT) was performed to measure the potency of immune serum. The HI titres of birds of groups A, B, D and E were 128, 16, 128 and 128 respectively after primary vaccination and 256, 128, 256 and 256 respectively after secondary vaccination. From the study it was found that among the experimental layer birds, no difference was noticed on the immune response to BCRDV. On the other hand, among the experimental broiler birds, birds of group A revealed higher HI titre compared to the birds of group B. Results of serum neutralization test indicated that the sera of birds possessing HI titre ?128 demonstrated a good level of virus neutralizing activity of ?7.60. The birds possessing HI titre of 128 ± 0.00 did not show any clinical signs of ND following challenge and the survivability was recorded as 100%. It may be concluded that supplementation of vitamin-mineral premix to the diet induces better immune response following vaccination with BCRDV in broiler chickens compared to the layer chickens.

Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

SummaryBacterial ghosts (BGs) have been applied through oral, aerogenic, intraocular or intranasal routes for mucosal immunization using a wide range of experimental animals. All these applications required a booster after primary immunization to achieve protective immunity against the lethal challenge. Here we report for the first time that a single rectal dose of BGs produced from enterohaemorrhagic Escherichia coli (EHEC) O157:H7 fully protects mice against a 50% lethal challenge with a heterologous EHEC strain given at day 55. BGs from EHEC O157:H7 were prepared by a combination of protein E‐mediated cell lysis and expression of staphylococcal nuclease A guaranteeing the complete degradation of pathogen residual DNA. The lack of genetic material in the EHEC BGs vaccine abolished any potential hazard for horizontal gene transfer of plasmid encoded antibiotic resistance genes or pathogenic islands to the recipient's gut flora. Single rectal immunization using EHEC O157:H7 BGs without any addition of adjuvant significantly stimulated efficient humoral and cellular immune responses, and was equally protective as two immunizations, which indicates the possibility to develop a novel efficacious single dose mucosal EHEC O157:H7 BGs vaccine using a simplified immunization regimen.

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS (1-4). This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells (5-12). The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site (6, 7, 13-15). Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve (16-19) or injecting vectors into their target the superior colliculus (10). This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Effect of Subconjunctival and Intraocular Bevacizumab Injections on Corneal Neovascularization in a Mouse Model

Purpose: This study sought to evaluate the efficacy of bevacizumab (Avastin®), an anti-VEGF agent, in the reduction of corneal neovascularization in a rodent model, and compare between the effect of intraocular and subconjunctival injection.Methods: Corneal neovascularization was induced by application of a mixture of 75% silver nitrate and 25% potassium nitrate to the corneal center of the right eye of 125 mice. Immediately thereafter, 75 eyes were treated with subconjunctival, anterior chamber, or intravitreal injection of bevacizumab. The remaining mice served as a control. The groups were compared at different postoperative time points for percentage area of neovascularization evaluated by digital photos, the presence of corneal stromal vascular endothelial cells studied by immunohistochemical staining, and VEGF levels measured by real-time PCR.Results: In the untreated eyes, percentage area of neovascularization increased from 11.3 ± 7% on day 2 to 20 ± 8.9% on day 4, 47 ± 25.4% on day 8, and 51 ± 24.7% on day 10. The percentage area of neovascularization following subconjunctival injection was 7 ± 2.9% on day 2, 15.7 ± 6% on day 4, 32.2 ± 15.2% on day 8, and 39.7 ± 14.5% on day 10. The percentage area of neovascularization following anterior chamber injection was 14.4 ± 3% on day 2, 16.5 ± 6.3% on day 4, 26.3 ± 6.5% on day 8, and 19.8 ± 1.2% on day 10. The percentage area of neovascularization following intravitreal injection was 11 ± 2.5% on day 2, 14.7 ± 5% on day 4, 23.5 ± 6.5% on day 8, and 24.2 ± 14.9% on day 10.Conclusions: Bevacizumab injections partially inhibit the growth of corneal neovascularization induced by acute chemical injury in a mouse model. The intraocular routes of injection were found to be the most effective, and the subconjunctival route of injection yielded the earliest peak response.

Susceptibility of Guinea Fowl (Numida Meleagris Galeata) to Infectious Bursal Disease Virus (IBDV)

The experiment was carried out to determine whether the guinea fowls are susceptible to Infectious Bursal Disease Virus (IBDV). A total of 60 guinea fowl keets were used for the study. They were administered the inoculum through the intraocular route. 30 guinea fowl keets served as the control group (A) while, the other 30 served as the experimentally infected group (B). The only clinical signs observed in about 10 keets were mild depression and transient loss of appetite that did not last more that 24 h. The morphometric observations also indicated that the relative weights in the two groups (A and B) showed no Significant difference (p>0.05) in both the Control (group A) and Infected (group B) keets. It was concluded that guinea fowls are not susceptible to IBDV but they could severe as carriers, which would spread the virus to other poultry species if reared together with them.

Evaluation of two scopolamine and physostigmine pretreatment regimens against nerve agent poisoning in the dog

Currently, there is no viable protection against chemical warfare agents for the working dog. Physostigmine (PHY) and scopolamine (SCO) have been shown to protect dogs against nerve agents with minimal side effects. The goal of this study was to investigate whether reported protective SCO/PHY plasma concentrations of 0.2 and 0.7 ng/mL, respectively, could be reached and maintained with minimal side-effects thereby identifying possible pretreatment regimens. Two continuous regimens of SCO/PHY were administered to beagle dogs. The first regimen consisted of administering transdermal SCO and intraocular PHY, the second consisted of transdermal SCO and rectal PHY. SCO/PHY plasma concentrations for each regimen were determined, individual protective times were calculated and a computerized pharmacokinetic analysis was performed. The results showed transdermal SCO and intraocular PHY routes of delivery achieved sustained protective drug concentrations with minimal side-effects and the rectal route of delivery did not. Group median protective times for the first regimen were 54.45 h for SCO and 64.35 h for PHY, and for the second regimen 63.75 h for SCO and 0 h for PHY. The combined transdermal patch and intraocular regimen may provide a safe and effective regimen against nerve agent poisoning in dogs.

Ocular surface-evoked Fos-like immunoreactivity is enhanced in trigeminal subnucleus caudalis by prior exposure to endotoxin

Endotoxin-induced uveitis (EIU) is a common animal model for anterior uveitis in humans that causes long-term changes in trigeminal brain stem neurons. This study used c-fos immunohistochemistry to assess the effects of different routes of administration of endotoxin on activation of trigeminal brain stem neurons produced by ocular surface stimulation. A single dose of endotoxin (lipopolysaccharide (LPS)) given to male rats by systemic (i.p., 1 mg/kg) or intraocular (ivt, 20 μg) routes increased the number of Fos-positive neurons in rostral (trigeminal subnucleus interpolaris/subnucleus transition (Vi/Vc)) and caudal portions of trigeminal subnucleus caudalis (trigeminal subnucleus caudalis/upper cervical spinal cord transition (Vc/C 1–2)) by 20% mustard oil (MO) applied to the ocular surface 7 days, but not at 2 days, after LPS compared with naïve rats. I.c.v. (20 μg) LPS did not affect MO-evoked Fos. To determine if the pattern of enhanced Fos expression after systemic LPS also depended on the nature of the ocular surface stimulus, additional groups received ocular stimulation by 10% histamine or dry eye conditions. Seven days, but not 2 days, after i.p. LPS both histamine- and dry eye–evoked Fos was increased at the Vi/Vc transition, while smaller effects were seen at other regions. These results suggested that EIU modulation of trigeminal brain stem neuron activity was mediated mainly by peripheral actions of LPS. Enhancement of Fos at the Vi/Vc region after MO, histamine and dry eye conditions supports the hypothesis that this region integrates innocuous as well as noxious sensory information, while more caudal portions of Vc process mainly nociceptive signals from the eye.

Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally

Asian H5N1 (hereafter referred to as panzootic H5N1) highly pathogenic avian influenza (HPAI) virus has caused large numbers of deaths in both poultry and wild-bird populations. Recent isolates of this virus have been reported to cause disease and death in commercial ducks, which has not been seen with other HPAI viruses. However, little is known about either the dissemination of this H5N1 within the organs or the cause of death in infected ducks. Nineteen 4-week-old Pekin ducks were infected with 106.7 median egg infectious doses of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2) in 0.1ml via the intranasal and intraocular routes. Cloacal and oropharyngeal swabs were taken daily before three animals were selected randomly and killed humanely for postmortem examination, when samples of tissues were taken for real-time reverse transcriptase-polymerase chain reaction, histopathological examination and immunohistochemistry. Clinical signs were first observed 4 days post infection (d.p.i.) and included depression, reluctance to feed, in-coordination and torticollis resulting in the death of all the birds remaining on 5d.p.i. Higher levels of virus shedding were detected from oropharyngeal swabs than from cloacal swabs. Real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry identified peak levels of virus at 2d.p.i. in several organs. In the spleen, lung, kidney, caecal tonsils, breast muscle and thigh muscle the levels were greatly reduced at 3d.p.i. However, the highest viral loads were detected in the heart and brain from 3d.p.i. and coincided with the appearance of clinical signs and death. Our experimental results demonstrate the systemic spread of this HPAI H5N1 virus in Pekin ducks, and the localization of virus in the brain and heart tissue preceding death.

Time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO 2 nanoparticles

Nanoparticles can be administered via nasal, oral, intraocular, intratracheal (pulmonary toxicity), tail vein and other routes. Here, we focus on the time-dependent translocation and potential damage of TiO 2 nanoparticles on central nervous system (CNS) through intranasal instillation. Size and structural properties are important to assess biological effects of TiO 2 nanoparticles. In present study, female mice were intranasally instilled with two types of well-characterized TiO 2 nanoparticles (i.e. 80 nm, rutile and 155 nm, anatase; purity > 99%) every other day. Pure water instilled mice were served as controls. The brain tissues were collected and evaluated for accumulation and distribution of TiO 2, histopathology, oxidative stress, and inflammatory markers at post-instillation time points of 2, 10, 20 and 30 days. The titanium contents in the sub-brain regions including olfactory bulb, cerebral cortex, hippocampus, and cerebellum were determined by inductively coupled plasma mass spectrometry (ICP-MS). Results indicated that the instilled TiO 2 directly entered the brain through olfactory bulb in the whole exposure period, especially deposited in the hippocampus region. After exposure for 30 days, the pathological changes were observed in the hippocampus and olfactory bulb using Nissl staining and transmission electron microscope. The oxidative damage expressed as lipid peroxidation increased significantly, in particular in the exposed group of anatase TiO 2 particles at 30 days postexposure. Exposure to anatase TiO 2 particles also produced higher inflammation responses, in association with the significantly increased tumor necrosis factor alpha (TNF-α) and interleukin (IL-1β) levels. We conclude that subtle differences in responses to anatase TiO 2 particles versus the rutile ones could be related to crystal structure. Thus, based on these results, rutile ultrafine-TiO 2 particles are expected to have a little lower risk potential for producing adverse effects on central nervous system. Although understanding the mechanisms requires further investigation, the present results suggest that we should pay attention to potential risk of occupational exposure for large-scaled production of TiO 2 nanoparticles.

Viral load in 1-day-old and 6-week-old chickens infected with chicken anaemia virus by the intraocular route

Although the effects of chicken anaemia virus (CAV) infection have frequently been investigated in young chickens, there have been few studies of the pathogenesis of CAV infection in older birds. The aim of the work reported here was to study viral loads in 6-week-old chickens and to compare these with those seen in younger birds. Specific pathogen free chickens were inoculated at 1 day or at 6 weeks of age with 10(4) median tissue culture infective doses of CAV by the intraocular route. Chicks infected when 1 day old were euthanized at day 14, 18 or 22 post inoculation (p.i.), and those infected when 6 weeks old at day 16, 18 or 20 p.i. Their body and thymus weights were determined and samples were collected from their spleen, liver and thymus. A quantitative polymerase chain reaction assay was developed and used to determine the number of viral genome copies in the tissue samples. In both age groups, viral genome concentrations increased in all organs up to day 18 p.i. and reached a peak in the spleen and liver at day 18 p.i. The peak viral concentrations in the thymus were detected at day 18 in the younger birds and at day 20 p.i. in older chickens. These studies have shown that exposure to CAV in older birds leads to similar levels of active viral replication to those seen in younger birds, and may result in subclinical infections in older birds with the potential to increase susceptibility to other infectious agents.