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Objective The current study aims to evaluate the utility of impression cytology in the diagnosis of ocular surface alterations in multiple sclerosis patients as well as to connect impression cytology results with symptoms and clinical evaluation data. Patients and Methods: The study included 60 patients [120 eyes] who were divided into 3 groups: Group 1: included 20 participants (40 eyes) with early-diagnosed multiple sclerosis; group 2: included 20 participants (40 eyes) with multiple sclerosis who are already receiving treatment; and group 3: included 20 participants (40 eyes) who were randomly assigned to an age-matched control group who had no ocular or systemic pathologies. In people aged 20 to 40, an ocular tear film was evaluated. All patients finished the routine ophthalmological assessment, fluorescein tear breakup time [TBUT], Schirmer test, and conjunctival impression cytology [CIC]. Results Most of the early group patients showed grades 1, 2 and 3 (20%, 35% and 30% respectively) in CIC results in 85% of the eyes, whilst in the chronic group patients showed grades 1, 2 and 3 (35%, 25%and 15% respectively) in CIC results in 75% of the eyes, while in the control subjects, impression cytology results showed that all cases were grade 0 (100%) with a highly statistically significant difference with P-value

Purpose The aim of this study was to evaluate and to compare the thickness and morphology of femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and mechanical microkeratome flaps using anterior segment optical coherence tomography (AS-OCT). Patients and methods This prospective, interventional, comparative clinical study was performed on 60 eyes of 30 patients. Flaps were created in 30 eyes using the WaveLight FS200 FS laser and in 30 eyes using the Moria M2 microkeratome. AS-OCT was used at 1 month postoperatively to evaluate the thickness of each flap at four points, which were 1 and 2 mm from the corneal vertex on the horizontal meridian. Results At the sixth month postoperatively, the uncorrected visual acuity was 0.98±0.10 in the FS-LASIK group and 0.98±0.11 in the microkeratome group, which denotes the efficacy of both procedures. No vision-threatening complications occurred in either group. The average flap thickness at 1 mm was 114.93±3.04 µm in the FS-LASIK group compared with 128.57±3.98 µm in the microkeratome group, with a statistically significant difference (P=0.000). The difference between the achieved and intended flap thickness at 1 mm (accuracy) was 4.93±3.04 µm in the FS-LASIK group compared with 18.57±3.98 µm in the microkeratome group, with a statistically significant difference (P=0.000). At 2 mm, the average flap thickness was 115.22±3.34 µm in the FS-LASIK group compared with 139.00±4.75 µm in the microkeratome group (P=0.000). The flap accuracy at 2 mm was 5.22±3.34 µm and 29.00±4.75 µm in the FS-LASIK group and the microkeratome group, respectively (P=0.000). Flap morphology showed a planar shape (uniform) in the FS-LASIK group and a meniscus shape in the microkeratome group. Conclusion Both FS-LASIK and microkeratome techniques were safe and efficient in correcting myopia and myopic astigmatism, achieving good visual outcome. AS-OCT showed that flaps created by the FS laser were more accurate and uniform than those created by the Moria M2 microkeratome.

Plant Growth Promoting Rhizobacteria (PGPR) were evaluated individually or in combination and compared to the fungicide Topsin M-70® for their antagonistic activities and suppressive actions against Fusarium oxysporum and Rhizoctonia solani severally, which were deemedthe causal agents of root rot and wilt diseases of faba bean plants. In vitro, most of the examined strains showed eminent abilities to inhibit mycelial growth of Rhizoctonia solani or Fusarium oxysporum. Strains of Rhizobium leguminosarum (No.9), Azotobacterchroococcum (No.12) and Pseudomonas fluorescens (No. 6) recorded the highest inhibition zone against two tested pathogenic fungi and were used in the present study. Generally, under greenhouse and field conditions, all the tested PGPR strains significantly reduced root-rot and wilt incidence and increased the percentage of survived plants compared to control (infested soil). Combination among R. leguminosarum+ A. chroococcum + P. fluorescens was more effective in controlling root-rot and wilt diseases, increasing growth parameters, yield components, photosynthetic pigments, activities of nitrogenase, dehydrogenase and total phenols. Obtained results clarified the importance of PGPR in controlling pathogenic fungi, enhancing growth parameters of faba been plants compared to chemical fungicide and lowering chemical fertilizers application.

Objective The aim of this study was to evaluate the corneal endothelial changes following corneal collagen cross-linking treatment of keratoconus using a specular microscope. Patients and methods The study included 30 eyes of 30 patients with keratoconus, comprsing 16 males and 14 females, with a mean age of 28 years. Patients had a standard corneal collagen cross-linking after removal of the corneal epithelium. They were evaluated before and 1 month after the procedure using the specular microscope. Results The mean preoperative central corneal thickness of the patients was 477.5±35.57 µm, and at 1 month postoperatively, it was 477.9±36.88 µm, with no statistically significant change (P=0.7821). The mean preoperative value of cell density was 2963.1±364.1 cells/mm2 and became 2956.96±363 cells/mm2 1 month postoperatively, with no statistically significant change (P=0.7091). The mean preoperative coefficient of variation value was 36.3±5.9%, whereas 1 month postoperatively, it became 36.2±5.6%, with no statistically significant change (P=0.6951). The mean preoperative value of hexagonal cells was 49.93±7.9% and became 48.5±7.3% 1 month postoperatively, with no statistically significant change (P=0.2078). Conclusion There were no significant changes in human corneal endothelial parameters, as evaluated with the specular microscope, following cross-linking in keratoconic eyes.

Background: A family history of glaucoma was found to carry a relative risk of 2.1 times for being associated with at least possible primary open angle glaucoma (POAG) in middle aged and older individuals. Around half of all POAG patients have a positive family history and their first degree relatives have an approximately 9-fold increased risk of developing glaucoma. Objective: the aim of the present study was to demonstrate possible early macular alterations in young relatives of POAG patients using spectral domain optical coherence tomography (SD-OCT) posterior pole asymmetry analysis (PPAA) map. Patients and methods: in a prospective comparative study; 40 subjects were classified into 2 groups: group of young healthy relatives of POAG patients and another group of young healthy subjects as control. All participants had OCT of the PPAA, retinal nerve fiber layer (RNFL), ganglion cell complex (GCC) and hemi-field macular thickness. Results: There was no statistically significant difference between the relative and control group in aspect of RNFL, macular hemi-fields and PPAA measurements (P-value >0.05). Regarding GCC thickness, there were statistically significant differences between both groups in the superior, inferior and nasal thickness measurements. Conclusion: Young healthy relatives of POAG patients didn’t show any early glaucomatous damage when compared with controls except for GCC thickness which showed statistically significant decrease.

Background: Chronic obstructive pulmonary disease (COPD) has multi-systemic implications and comorbidities. Fine ocular structures are affected by hypoxia and systemic inflammation. Objective: To evaluate the changes in the retina and choroid in patients with chronic obstructive pulmonary disease using spectral-domain optical coherence tomography (SD-OCT). Patients and methods: This prospective case control study included a total of forty COPD patients and twenty ageand sex-matched healthy individuals as control group; both eyes were included. COPD patients were divided into two subgroups; a group with mild to moderate COPD and another group with severe to very severe COPD according to the Global Initiative for COPD guideline. COPD patients were subjected to spirometry. Arterial blood gas analysis was performed to determine hypoxic state. The subfoveal choroidal thickness (SFCT), macular thickness (MT) and retinal nerve fiber layer (RNFL) thickness were measured using SD-OCT and compared between groups. Results: There was no statistically significant difference between groups according to demographic data. Mean SFCT was statistically significantly thinner in COPD groupsthan in the control group; and this thinning was more pronounced with increase in the severity of COPD. As regard to MT, no statistically significant difference was observed between groups (p=0.691). Peripapillary RNFL was significantly thinner in COPD group than the control group in all quadrants except the superior one. Conclusion: Changes in the SFCT and RNFL thickness could be used as indicators for the severity of COPD

This case represents an isolated cavernous hemangioma of the optic disc diagnosed by various diagnostic modalities. A 32-year-old woman with no ocular or systemic history was diagnosed with cavernous hemangioma of the optic disc. Fundus examination revealed a cluster of grape-like lesion occupying the lower part of the optic disc and the adjacent peripapillary retina. Fundus fluorescein angiography showed delayed filling of the lesion with late intravascular plasma–erythrocyte separation ‘characteristic fluorescein capping’ with no leakage. Optical coherence tomography revealed dilated saccular clusters occupying the lower optic disc and adjacent retina. Optical coherence tomography angiography showed a hyperintense lesion (denoting vascular flow) involving the lower half of the disc. No associated systemic hemangiomas were found. After a 1-year follow-up period, no changes have been documented.

Highly pathogenic avian influenza subtype H5N1 represents a threat to the poultry industry and human health worldwide. Inapparently infected birds are suspected to play an essential role in the spread of avian influenza virus. In the current study, a total of 25,646 samples (16,185 chicken, 4696 ducks, 1633 geese and 3132 turkeys) from apparently healthy birds were screened for the presence of positive samples for H5N1 during 2009-2014. The samples were examined by reverse transcriptase real-time polymerase chain reaction (rRT-PCR) for M, H5 and N1 genes of avian influenza viruses. The results revealed that the HPAI H5N1 existed in an inapparent manner in ducks (4.68 %), geese (4.10 %), chickens (2.48 %) and turkeys (2.29 %). The current finding highlights the serious impact of such type on birds in the epidemiology of H5N1 in birds, animals and humans. It also highlights the existence of another reason other than vaccination that contributes to the widespread of inapparent infection of H5N1 in Egypt.

A total 2565 samples (1890 samples of frozen chicken, 660 samples of freshly slaughter chicken were collected from neck skin, cloacal skin and drip and 15 samples of washing containers) from super market and slaughter poultry house in Egypt and examined for presence of Campylobacter. The Campylobacter was detected by 16.7 % of all examined samples. Incidence of Campylobacter in frozen samples was 7.94 %, 1.59 %, 9.0 % in neck skin, cloacal skin and drip respectively. The results freshly slaughter chicken of revealed 32.73 % incidence of Campylobacter in samples. The incidence of Campylobacter in the examined washing water were 100%. The cross contamination was occurred during the slaughter processing. And Continues test of poultry carcasses and by-product before packing and distribution is highly recommended with application of good hygienic measure importance to reduce human infection.

Purpose The aim of this study was to evaluate the effect of preoperative subconjunctival bevacizumab injection on the clinical and immunohistochemical analysis of primary pterygium. Patients and methods A total of 10 eyes of 10 patients with primary pterygium received a subconjunctival injection of bevacizumab (2.5 mg/0.1 ml) 1 week before simple pterygium excision with bare sclera technique. The control group (10 eyes of 10 patients with primary pterygium) had simple excision of primary pterygium with bare sclera without prior injection. Follow-up was performed on the first week, 2 weeks, 1 month, and 3 months postoperatively to evaluate the surgical site vascularization and occurrence of any complications and to record the rate of recurrence. Immunohistochemical analysis was performed on the excised tissue to detect vascular endothelial growth factor (VEGF) in both groups. Results There was a marked decrease in pterygium vascularity after intralesional injection of bevacizumab. The injection group showed no recurrence after excision of pterygium during the scheduled follow-up period without any recorded complications regarding the use of bevacizumab, whereas the control group showed recurrence of pterygium in three eyes. The immunohistochemical analysis showed lower VEGF in the injected group than in the control group. Conclusion Preoperative injection of bevacizumab effectively reduced vascularity and VEGF concentration of pterygium tissue. A subconjunctival preoperative single dose of bevacizumab was not associated with any adverse effects and was well tolerated. It had a minimizing effect on the extent of vascularization of the pterygium. No recurrence of pterygium was noted in any of the injected patients.

Serologic tests are a valuable tool for retrospective surveillance of avian influenza viruses (AIV) and monitoring of postvaccination host immune response. Yet collection of serum samples, particularly in adult breeder chickens, is laborious, intrusive to birds, and may pose a serious risk to the biosecurity of a flock. In this study we compared the level of AIV-specific antibody titers in eggs and serum samples obtained from broiler breeder chickens vaccinated at 6, 12, and 18 wk of age with H5N2-inactivated vaccine. Nucleocapsid protein-specific ELISA and hemagglutination inhibition test (HI) against homologous as well as heterologous antigens were used. The eggs and sera were collected at 22, 30, 45, and 50 wk of age (i.e., 4, 12, 27, and 32 wk after the third and final immunization, respectively). Using ELISA, the number of positive egg yolk samples decreased over time after vaccination, from 97% to 47%, while the seropositivity rate of serum samples was 97%-100% during the whole investigation period. No antibody titers were detected in egg white. By HI, antibody titers in serum samples were higher than in egg yolk samples. Compared to the homologous H5N2 antigen, significantly lower HI titers were obtained by using a heterologous H5N1 virus of clade 2.2.1.2. In addition, no HI titers were detected in egg yolk and/or serum samples tested against the antigen of an Egyptian H5N1 antigenic drift variant of clade 2.2.1.1. This study indicates that egg yolk may be used to monitor the postvaccination immune status of broiler breeder chickens and retrospective serosurveillance-by HI when a matching antigen is available as well as by ELISA-particularly for up to 12 wk postvaccination.

Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was first diagnosed in poultry in Egypt in 2006, and since then the disease became enzootic in poultry throughout the country, affecting the poultry industry and village poultry as well as infecting humans. Vaccination has been used as a part of the control strategy to help to control the disease. Epidemiological data with sequence analysis of H5N1 viruses is important to link the mechanism of virus evolution in Egypt. This study describes the evolutionary pattern of Egyptian H5N1 viruses based on molecular characterization for the isolates collected from commercial poultry farms and village poultry from 2006 to 2011. Genetic analysis of the hemagglutinin (HA) gene was done by sequencing of the full-length H5 gene. The epidemiological pattern of disease outbreaks in Egyptian poultry farms seems to be seasonal with no specific geographic distribution across the country. The molecular epidemiological data revealed that there are two major groups of viruses: the classic group of subclade 2.2.1 and a variant group of 2.2.1.1. The classic group is prevailing mainly in village poultry and had fewer mutations compared to the originally introduced virus in 2006. Since 2009, this group has started to be transmitted back to commercial sectors. The variant group emerged by late 2007, was prevalent mainly in vaccinated commercial poultry, mutated continuously at a higher rate until 2010, and started to decline in 2011. Genetic analysis of the neuraminidase (NA) gene and the other six internal genes indicates a grouping of the Egyptian viruses similar to that obtained using the HA gene, with no obvious reassortments. The results of this study indicate that HPAI-H5N1 viruses are progressively evolving and adapting in Egypt and continue to acquire new mutations every season.

This study describes the first isolation of H9N2 avian influenza virus (AIV) from commercial bobwhite quail (Colinus virginianus) in Egypt. Infected birds showed neither clinical signs nor mortality. Virus isolation and real-time reverse transcription polymerase chain reaction confirmed the presence of the H9N2 virus in cloacal swab samples collected at 35 days of age and the absence of other AIV subtypes, including H5 and H7. The hemagglutinin and neuraminidase genes of the isolated virus showed 99.1% and 98.2% nucleotide identity and 97.3% and 100% amino acid identity, respectively, to those of H9N2 viruses currently circulating in poultry in the Middle East. Phylogenetically, the Egyptian H9N2 virus was closely related to viruses of the G1-like lineage isolated from neighbouring countries, indicating possible epidemiological links.

SUMMARY The lack of biosecurity measures for backyard birds in Egypt and their continuous contact with wild migratory birds make backyard birds much more vulnerable to avian influenza virus (AIV) than those on commercial farms. The wild bird-backyard flock pathway was claimed to be the source of the first intrusion of H5N1 virus in Egypt in February 2006. The Egyptian government reported the isolation of low-pathogenic AIV subtype H7 in migratory ducks from El-Abassa Lake, a fish-farming lake located 80 km east of Cairo. Fear of repeating the scenario of introducing H5N1 into the poultry sectors prompted us to survey backyard birds around the borders of the lake. Screening of 207 serum samples (73 from chickens, 67 from ducks, 52 from geese, 9 from turkeys, and 6 from pigeons) using a commercial generic anti-influenza A nucleoprotein antibody enzyme-linked immunosorbent assay revealed 4.9% positive chicken sera. No antibody against the AIV H7 subtype was recorded when a hemagglutination inhibition test was used, whereas 4.3% of the serum samples examined were positive for the H5 AIV subtype. Nevertheless, AIV was not detected in swabs examined by real-time reverse transcription-polymerase chain reaction. However, continuous surveillance of backyard birds should be emphasized.

SUMMARY Mycoplasma gallisepticum (MG) infections cause significant economic losses to the poultry industry worldwide. Vaccination with live and inactivated vaccines and administration of antibiotics are commonly used as control measures. Herein, we describe a 27-wk-old broiler-breeder vaccinated with live TS-11 MG vaccine at 10 wk of age. The flock subsequently exhibited mild respiratory signs and a slight decrease in egg production at 27 wk of age. Serological examination of serum samples using a serum plate agglutination test revealed 32% positive samples 6 wk after vaccination and 100% positive samples after the onset of clinical disorders. Detection of MG by polymerase chain reaction and partial sequencing of the second cytadhesin-like protein encoding gene (mgc2) showed 99% nucleotide sequence identity with MG field strains and a lower identity with the TS-11 vaccine strain. We conclude that vaccination of chickens with TS-11 against MG will not prevent infection with field strains. The serum plate agglutination test could be used to indicate field infection in flocks vaccinated with TS-11. However, the molecular detection and differentiation of MG strains are essential in controlling MG infection in poultry.

Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector.

The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., "variant" (clade 2.2.1var) and "proper" (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns.

In this study, we have compared the protection afforded by a recombinant turkey herpesvirus vaccine expressing the H5 gene from a clade 2.2 H5N1 strain (rHVT-H5) and a Mexican-origin H5N2 inactivated vaccine, alone or in combination, against two antigenically divergent H5N1 Egyptian strains isolated in 2007 and 2008. Our results confirm the existence of a major antigenic drift among the Egyptian H5N1 strains such that, although protection against the "classical" 2007 HPAI H5N1 Egyptian strain could be obtained with both types of vaccines, only vaccination with the rHVT-H5 vaccine protected against challenge with the "variant" 2008 HPAI H5N1 Egyptian strain.

The efforts exerted to prevent circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in birds are the best way to prevent the emergence of a new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry has increased since late 2007 as a result of the presence of genetic and antigenic distinct variant strains that have escaped during the immune response of vaccinated birds. Although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1-infected birds, escape of variant strains from detection by real-time reverse transcriptase-PCR (RRT-PCR) was observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target sequences of the H5 gene by real-time RT-PCR. This study describes the results of RRT-PCR, modified from an existing protocol with regard to the detection of the partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied to nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in the detection of Egyptian isolates, with 104% amplification efficiency. Sixty-one field samples were found to be positive in our assay, but only 51 samples tested positive by the original protocol and were more sensitive than matrix gene RRT-PCR detection assay. A detection limit of 10 mean embryo infective dose (EID50) with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid in the containment of the H5N1 virus in poultry sectors and to lower the threat of influenza virus spread.

The poultry meat trade in Egypt depends mainly on live bird markets (LBMs) because of insufficient slaughterhouses, lack of marketing infrastructure, and cultural preference for consumption of freshly slaughtered poultry. There are two types of LBMs in Egypt: retail shops and traditional LBMs where minimal, if any, food safety standards or veterinary inspection are implemented. Before January 2009, LBMs were considered to be a missing link in the epidemiology of avian influenza in Egypt. This incited us to initiate this surveillance to better understand the perpetuation of H5N1 and the risk of infection in poultry markets. Seventy-one out of 573 (12.4%) examined LBMs were positive for the H5N1 subtype by real-time--quantitative polymerase chain reaction (RT-qPCR) from January to April 2009. Where a 70.4% detection rate from LBMs had waterfowl only as a solitary sold species, a 26.8% detection rate from LBMs had waterfowl mixed with chicken and/or turkey, and 2.8% from LBMs had only turkey. Higher incidence, 40.8%, of positive LBMs was recorded during the cold month of February and concentrated mainly in the highly populated Nile Delta. These findings revealed wide circulation of H5N1 avian influenza virus in LBMs in Egypt, which poses a threat to public health and the poultry industry. Long-term control measures are required, and routine surveillance of bird markets should be conducted year-round.

The highly pathogenic avian influenza virus of subtype H5N1 that caused serious outbreaks in Egypt in 2006 was efficiently detected using a commercially available real-time reverse transcriptase-PCR (RRT-PCR) for the type A specific matrix (M) gene in field samples of cloacal and tracheal swabs. RRT-PCR was also used for subtyping and confirmation of H5 subtype. During late 2007 the National Laboratory for Veterinary Quality Control on Poultry Production detected five field cases that were positive for avian influenza virus (AIV) based on the M gene RRT-PCR. Three different commercial H5 RRT-PCRs were used for identification of the H5 subtype, as well as a published World Organization for Animal Health (OIE) H5 RRT-PCR that had been previously carefully validated. The five cases had positive results for the H5 gene using the published OIE H5 RRT-PCR, but the three commercial H5 RRT-PCRs tests only returned two to four positive results out of the five positive cases. The hemagglutinin gene (HA) sequencing analysis of these five isolates showed multiple nucleotide substitution mutations, suggesting genetic variation that could affect the H5 primer and/or probe binding sequences. These data highlight the importance of continued monitoring of RRT-PCR primers and probes to ensure that sensitivity and specificity are maintained. The use of conventional methods in national and reference AIV laboratories, including virus isolation, serologic subtyping, and alternative RRT-PCR primers, is necessary to detect the newly emerging variant H5N1 strains that affect diagnostic performance.

The Eurasian-lineage H5N1 highly pathogenic avian influenza (HPAI) virus caused widespread outbreaks in Egypt in 2006 and eventually become enzootic in poultry. Although outbreaks have a seasonal pattern, with most occurring during the cooler winter months, it remains unclear whether this seasonality reflects virus maintenance within Egypt or yearly introductions of the virus into the country. To evaluate the epidemiology of H5N1 HPAI in Egypt, sequence analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of selected Egyptian isolates from early 2006 to 2008 was conducted. The data from this study identifies distinct genetic markers in both HA and NA genes and suggests grouping Egyptian isolates into two major HA isolate sublineages from 2006 to 2008 and into three smaller, emergent subgroups. The NA phylogenetic and sequence analysis showed a similar pattern, except that two of the emergent groups from the HA phylogenetic tree clustered together, evidence of likely reassortment. The different subgroups did not appear to segregate by relation to the date of isolation, to the species of origin, nor to the geographic location of the viruses. The conclusion is that H5N1 is continuing to mutate with multiple heterogenic strains persisting in Egypt.

Although linked, these viruses are distinct from earlier outbreak strains., To better understand the ecology and epidemiology of the highly pathogenic avian influenza virus in its transcontinental spread, we sequenced and analyzed the complete genomes of 36 recent influenza A (H5N1) viruses collected from birds in Europe, northern Africa, and southeastern Asia. These sequences, among the first complete genomes of influenza (H5N1) viruses outside Asia, clearly depict the lineages now infecting wild and domestic birds in Europe and Africa and show the relationships among these isolates and other strains affecting both birds and humans. The isolates fall into 3 distinct lineages, 1 of which contains all known non-Asian isolates. This new Euro-African lineage, which was the cause of several recent (2006) fatal human infections in Egypt and Iraq, has been introduced at least 3 times into the European-African region and has split into 3 distinct, independently evolving sublineages. One isolate provides evidence that 2 of these sublineages have recently reassorted.

The responses of vaccinated and unvaccinated chickens of different breeds to infection with very virulent infectious bursal disease virus (vvIBDV) were investigated. Five-week-old chickens of five Egyptian breeds (Fayoumi, Balady, Golden, Mandarah, and Gimmizah), and foreign White Leghorn pullets were tested. In unvaccinated birds, mortality, relative bursa and spleen weight, bursal lesion score, antibody titres and the response of blood lymphocytes to mitogens were examined. The Gimmizah and Fayoumi birds showed the greatest susceptibility to infection with mortalities of 85 and 47%, respectively. Mandarah birds were resistant (11% mortality), while the Leghorn, Golden and Balady birds were of intermediate susceptibility (20, 21 and 37% mortality rates, respectively). Vaccinated birds were administered a live intermediate classic vaccine and challenged 10 days later with vvIBDV. Mortality following challenge was about 3% in the Fayoumi, Gimmizah and Balady birds, whereas no mortality was seen in Mandarah, Golden, and Leghorn birds. The classic vaccine induced significant protection. However, it did not prevent histological bursal lesions, especially in the Fayoumi and Leghorn birds. Neither pathological nor immunological measures correlated closely with susceptibility or resistance of the different breeds. The findings suggest that innate non-immunogenic factor(s) may play a critical role in resistance.

Purpose: To measure erythropoietin (EPO) level in vitreous and serum of patients with proliferative diabetic retinopathy (PDR) and to detect EPO single nucleotide polymorphism (SNP) genotyping expression in them. Materials and Methods: Concentrations of EPO in vitreous and serum of twenty diabetic patients with PDR undergoing vitrectomy (PDR group) were measured by chemiluminescent immunoassay and compared with those in nondiabetic (ND) patients undergoing vitrectomy for recent retinal detachment (ND group). Genotyping of EPO gene SNP (rs551238) was performed using real-time polymerase chain reaction technique. Results: There were a significant increase in serum EPO level in PDR group than in ND group and a highly significant increase in vitreous EPO level in PDR group than in ND group. Within the PDR group, vitreous EPO level was significantly higher than its serum level; there was up to 6-fold increase in the EPO vitreous level than its serum level. EPO gene SNP rs551238 was significantly associated with PDR in Egyptian patients. Conclusion: The results of the current study suggest the involvement of EPO in the pathogenesis of PDR. Hence, augmenting vascular endothelial growth factor inhibition with the use of EPO inhibitors may provide a better outcome in PDR treatment. The detection of EPO gene SNP rs551238 in Egyptian patients with PDR may help in the identification of the genetic predisposition to diabetic retinopathy in Egyptians and so may help in prevention and early detection of susceptible candidates to PDR.

Highly pathogenic avian influenza H5N1 virus (HPAIV) continues to be a candidate of a further influenza virus pandemic. Egypt is the country worst affected by human cases of HPAIV H5N1 infection in 2009. Increased infection of preschool children and decreased mortality rates suggested subtle changes in the epidemiology of the infection. Among other factors, the evolution of several conspicuous viral genetic markers in the HA and NA genes of HPAIV H5N1 viruses of human cases from Egypt and their putative influence on biological virus characteristics described here may contribute to this situation.

Background The endemic H5N1 high pathogenicity avian influenza virus (A/H5N1) in poultry in Egypt continues to cause heavy losses in poultry and poses a significant threat to human health. Methods Here we describe results of A/H5N1 surveillance in domestic poultry in 2009 and wild birds in 2009–2010. Tracheal and cloacal swabs were collected from domestic poultry from 22024 commercial farms, 1435 backyards and 944 live bird markets (LBMs) as well as from 1297 wild birds representing 28 different types of migratory birds. Viral RNA was extracted from a mix of tracheal and cloacal swabs media. Matrix gene of avian influenza type A virus was detected using specific real-time reverse-transcription polymerase chain reaction (RT-qPCR) and positive samples were tested by RT-qPCR for simultaneous detection of the H5 and N1 genes. Results In this surveillance, A/H5N1 was detected from 0.1% (n = 23/) of examined commercial poultry farms, 10.5% (n = 151) of backyard birds and 11.4% (n = 108) of LBMs but no wild bird tested positive for A/H5N1. The virus was detected from domestic poultry year-round with higher incidence in the warmer months of summer and spring particularly in backyard birds. Outbreaks were recorded mostly in Lower Egypt where 95.7% (n = 22), 68.9% (n = 104) and 52.8% (n = 57) of positive commercial farms, backyards and LBMs were detected, respectively. Higher prevalence (56%, n = 85) was reported in backyards that had mixed chickens and waterfowl together in the same vicinity and LBMs that had waterfowl (76%, n = 82). Conclusion Our findings indicated broad circulation of the endemic A/H5N1 among poultry in 2009 in Egypt. In addition, the epidemiology of A/H5N1 has changed over time with outbreaks occurring in the warmer months of the year. Backyard waterfowl may play a role as a reservoir and/or source of A/H5N1 particularly in LBMs. The virus has been established in poultry in the Nile Delta where major metropolitan areas, dense human population and poultry stocks are concentrated. Continuous surveillance, tracing the source of live birds in the markets and integration of multifaceted strategies and global collaboration are needed to control the spread of the virus in Egypt.

An evolutionary analysis was conducted of 354 hemagglutinin (HA) and 208 neuraminidase (NA) genes, including newly generated sequences of 5 HA and 30 NA, of Egyptian H5N1 clade 2.2.1 viruses isolated from poultry and humans. Five distinct phylogenetically distinguishable clusters arose from a monophyletic origin since 2006. Only two clusters remained in circulation after 2009: (i) A cluster of viruses arose in 2007 in industrial-vaccinated chickens and carried multiple mutations in or adjacent to the immunogenic epitopes of the HA. Viruses within this cluster evolved with significantly elevated mutation rates indicating persisting selective pressures, e.g. to escape host immunity and (ii) The second group arose in 2008 and harboured strains from recent human infections featuring a conspicuous deletion in the HA receptor-binding domain and substitutions close to the highly conserved active site of the NA. In both sublineages, a number of positively selected amino acids, different glycosylation patterns and variations in the polybasic proteolytic cleavage site were observed. Continuous monitoring of the evolving H5N1 virus in Egypt is essential to develop new control campaigns in poultry and human population.

Avian influenza due to highly pathogenic avian influenza (HPAIV) H5N1 virus is not a food-borne illness but a serious panzootic disease with the potential to be pandemic. In this study, broiler chickens were vaccinated with commercial H5N1 or H5N2 inactivated vaccines prior to being challenged with an HPAIV H5N1 (clade 2.2.1 classic) virus. Challenged and non-challenged vaccinated chickens were kept together, and unvaccinated chickens served as contact groups. Post-challenge samples from skin and edible internal organs were collected from dead and sacrificed (after a 14-day observation period) birds and tested using qRT-PCR for virus detection and quantification. H5N1 vaccine protected chickens against morbidity, mortality and transmission. Virus RNA was not detected in the meat or edible organs of chickens vaccinated with H5N1 vaccine. Conversely, H5N2 vaccine did not confer clinical protection, and a significant virus load was detected in the meat and internal organs. Phylogenetic analysis showed that the H5N1 virus vaccine and challenge virus strains are closely related. The results of the present study strongly suggest a need for proper selection of vaccines and their routine evaluation against newly emergent field viruses. These actions will help to reduce human exposure to HPAIV H5N1 virus from both infected live birds and slaughtered poultry. In addition, rigorous preventive measures should be put in place in order to minimize the public-health risks of avian influenza at the human-animal interface.

In Egypt, continuous circulation of highly pathogenic avian influenza (HPAI) H5N1 viruses of clade 2.2.1 in vaccinated commercial poultry challenges strenuous control efforts. Here, vaccine-derived maternal AIV H5 specific immunity in one-day old chicks was investigated as a factor of vaccine failure in long-term blanket vaccination campaigns in broiler chickens. H5 seropositive one-day old chicks were derived from breeders repeatedly immunized with a commercial inactivated vaccine based on the Potsdam/H5N2 strain. When challenged using the antigenically related HPAIV strain Italy/98 (H5N2) clinical protection was achieved until at least 10 days post-hatch although virus replication was not fully suppressed. No protection at all was observed against the Egyptian HPAIV strain EGYvar/H5N1 representing a vaccine escape lineage. Other groups of chicks with maternal immunity were vaccinated once at 3 or 14 days of age using either the Potsdam/H5N2 vaccine or a vaccine based on EGYvar/H5N1. At day 35 of age these chicks were challenged with the Egyptian HPAIV strain EGYcls/H5N1 which co-circulates with EGYvar/H5N1 but does not represent an antigenic drift variant. The Potsdam/H5N2 vaccinated groups were not protected against EGYcls/H5N1 infection while, in contrast, the EGYvar/H5N1 vaccinated chicks withstand challenge with EGYvar/H5N1 infection. In addition, the results showed that maternal antibodies could interfere with the immune response when a homologous vaccine strain was used.

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

Background The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites. Results We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt. Conclusions The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt.

In contrast to chickens, there is a paucity of information on the potency of H5 vaccines to protect turkeys against the highly pathogenic avian influenza (HPAI) H5N1 virus infections. In this study, 4 groups, 10 turkey poults each, were vaccinated at seven days old with one of H5N2 or H5N1 commercial vaccines or one of two prepared H5N1 vaccines from a local Egyptian variant HPAI H5N1 (EGYvar/H5N1) strain. At 35 days age, all vaccinated and 10 non vaccinated birds were challenged intranasal with 106 EID50/0.1 ml of EGYvar/H5N1. All vaccines used in this study were immunogenic in turkeys. There was no cross reaction between the commercial vaccines and the Egyptian variant H5N1 antigen as obtained by the hemagglutination inhibition test. Birds vaccinated with H5N2 vaccine were died, while other H5N1 vaccinated groups have had 20–40% mortality. The highest virus excretion was found in non-vaccinated infected and H5N2 vaccinated birds. Eleven peculiar amino acid substitutions in H5 protein of the variant strain were existed neither in the vaccine strains nor in the earliest H5N1 virus introduced into Egypt in 2006. In conclusion, single vaccination at seven days old is inadequate for protection of meat turkeys against variant HPAI H5N1 challenge and multi-dose vaccination at older age is recommended. For the foreseeable future, continuous evaluation of the current vaccines in H5N1 endemic countries in the face of virus evolution is a paramount challenge to mitigate the socio-economic impact of the virus.

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.

In this paper, we describe results from a high-pathogenic H5N1 avian influenza virus (AIV) surveillance program in previously H5-vaccinated commercial and family-backyard poultry flocks that was conducted from 2007 to 2008 by the Egyptian National Laboratory for Veterinary Quality Control on Poultry Production. The real-time reverse transcription PCR assay was used to detect the influenza A virus matrix gene and detection of the H5 and N1 subtypes was accomplished using a commercially available kit real-time reverse transcription PCR assay. The virus was detected in 35/3,610 (0.97%) and 27/8,682 (0.31%) of examined commercial poultry farms and 246/816 (30%) and 89/1,723 (5.2%) of backyard flocks in 2007 and 2008, respectively. Positive flocks were identified throughout the year, with the highest frequencies occurring during the winter months. Anti-H5 serum antibody titers in selected commercial poultry ranged from

Avian influenza is endemic in some species of wild birds and is generally believed to cause only an asymptomatic infection. These viruses are routinely transmitted from this wild bird reservoir to poultry in many areas all over the world. Low pathogenic avian influenza (LPAI) was previously reported in Egypt from different types of wild birds. This report describes the isolation and genetic characterization of H7N7 LPAI virus from a black kite (Milvus migrans), the first reported from this species, during surveillance done on wild birds in 2005. The black kite is a migratory bird that has breeding habitat in Europe and migrates in the winter to North Africa and the Middle East. Eight samples were collected in South Sinai, Egypt, and tested by virus isolation in embryonating chicken eggs. One sample had positive hemagglutination activity after the second passage in specific-pathogen-free embryos. Virus identification and characterization were done and the isolate was confirmed as H7N7 LPAI. The sequence data showed that this isolate was most closely related to European H7 strains isolated from domestic and wild birds.

Quickvue and Anigen lateral flow devices (LFDs) were evaluated for detection of H5N1 highly pathogenic avian influenza (HPAI) infections in Egyptian poultry. Sixty five chickens and two turkeys were sampled in eight flocks where H5N1 HPAI infection was suspected. Swabs (tracheal and cloacal) and feathers were collected from each bird for flockside testing by the two LFDs. The same clinical specimens were transported for laboratory testing by M gene RRT PCR where a positive result by this “gold standard” test for one or both swabs from a given bird indicated infection at the bird level, showing 57 birds (including 15 carcassess) to be truly AI infected. Among these 57, similar bird-level LFD testing of swabs showed 43 and 44 to be AI infected by Quickvue and Anigen LFDs, respectively. Nine birds were AI negative by M gene RRT PCR and both LFDs, and one was M gene RRT PCR negative but positive by both LFDs, suggesting one false positive LFD result. Sensitivities of the LFDs relative to M gene RRT PCR were 77.2% for Anigen and 75.4% for Quickvue tests, with 90.0% specificity for both. By including feathers with swabs for LFD testing, the number of LFD positives among 57 infected birds increased by four to 48 by Anigen and 47 by Quickvue, increasing the sensitivity of the LFDs to 84.2% and 82.5% for Anigen and Quickvue, respectively. Although LFD sensitivity cannot compare to the high sensitivity displayed by validated AI RRT PCRs, they may be utilised for flockside testing of birds infected with HPAI at the peak of viral shedding, when birds are displaying advanced clinical signs or sampled as fresh carcasses. Swabs are classic field specimens collected from outbreaks, but inclusion of feathers from birds infected with H5N1 HPAI increased LFD sensitivity. However, the LFD false positive observation emphasises the importance of returning samples for confirmatory laboratory testing.

The occurrence of Salmonella in 750 birds was assessed. The samples included the internal organs (caecal pouches, yolk sac, liver, and lung) of imported 1-day-old chicks (n = 150), grandparent chicks (n = 150), breeder chicks (n = 150), ducklings (n = 150), and turkey poults (n = 150), and paper-lined boxes (n = 250). Salmonellae isolated from the internal organs and paper-lined box of 1-day-old chicks, ducklings, and poults were mostly evident from the paper-lined box followed by caecal samples. Imported 1-day-old grandparent flocks were Salmonella free. Although 23.3% of the imported breeder flocks were positive for Salmonella, the imported duckling flocks and day-old turkey poults exhibited 19.3% and 12.6%, respectively. The widest diversity in isolated salmonellae was from the 1-day-old chicks where Salmonella Newport, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Shubra, Salmonella Saintpaul, and Salmonella Agona were isolated. On the other hand, two Salmonella serovars were isolated from the imported breeders, Salmonella Shubra and Salmonella Shipley, and from the imported ducklings, Salmonella Shubra and Salmonella Saintpaul. The three Salmonella serovars isolated from the imported day-old turkey poults were Salmonella Shubra, Salmonella Newport, and Salmonella Saintpaul. The high percentage and diversity of Salmonella isolation from the imported birds cause concern because of the zoonotic potential of this agent and its economical importance to the local commercial poultry breeding industry. From 80 samples investigated for Salmonella, the positivity of the standard microbiological technique method was 17.5% and of the polymerase chain reaction method (Salmonella-specific invA gene) was 22.5%. The concordance between the two methods was 90% (k = 0.850). Our results indicated that the polymerase chain reaction approach is better than culturing for detecting Salmonella in poultry samples when using the preenriched medium combinations used in this study.

Highly pathogenic avian influenza virus A/H5N1 was first officially reported in Africa in early 2006. Since the first outbreak in Nigeria, this virus spread rapidly to other African countries. From its emergence to early 2008, 11 African countries experienced A/H5N1 outbreaks in poultry and human cases were also reported in three of these countries. At present, little is known of the epidemiology and molecular evolution of A/H5N1 viruses in Africa. We have generated 494 full gene sequences from 67 African isolates and applied molecular analysis tools to a total of 1,152 A/H5N1 sequences obtained from viruses isolated in Africa, Europe and the Middle East between 2006 and early 2008. Detailed phylogenetic analyses of the 8 gene viral segments confirmed that 3 distinct sublineages were introduced, which have persisted and spread across the continent over this 2-year period. Additionally, our molecular epidemiological studies highlighted the association between genetic clustering and area of origin in a majority of cases. Molecular signatures unique to strains isolated in selected areas also gave us a clearer picture of the spread of A/H5N1 viruses across the continent. Mutations described as typical of human influenza viruses in the genes coding for internal proteins or associated with host adaptation and increased resistance to antiviral drugs have also been detected in the genes coding for transmembrane proteins. These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent. They further stress how imperative it is to implement sustainable control strategies to improve animal and public health at a global level.

Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province in 1996, followed by human cases in Hong Kong in 1997 1,2. The number of confirmed human cases now exceeds 300 and the associated Case Fatality Rate exceeds 60% 3. The genetic diversity of the serotype continues to increase. Four distinct clades or sub-clades have been linked to human cases 4-7. The gradual genetic changes identified in the sub-clades have been attributed to copy errors by viral encoded polymerases that lack an editing function, thereby resulting in antigenic drift 8. We report here the concurrent acquisition of the same polymorphism by multiple, genetically distinct, clade 2.2 sub-clades in Egypt, Russia, Kuwait, and Ghana. These changes are not easily explained by the current theory of “random mutation” through copy error, and are more easily explained by recombination with a common source. The recombination role is further supported by the high fidelity replication in swine influenza 9 and aggregation of single nucleotide polymorphisms in H5N1 clade 2.2 hemagglutinin 10.