Genetic characterization of newly emerging avian reovirus variants in chickens with viral arthritis/tenosynovitis in Israel.

In recent years, new avian reovirus (ARV) variants caused a variety of symptoms in chickens worldwide, the most important of which was Viral arthritis/tenosynovitis which caused substantial economic losses and has become a concern to the worldwide chicken industry. In this study, we characterized emerging ARV variants in Israel and analyzed their genetic relationship with reference strains. One hundred thirty-four ARV variants were isolated from tendons and synovial fluids of commercial broiler chickens with signs of arthritis/tenosynovitis. Phylogenetic analysis of the partial segment of the sigma C (σC) gene confirmed that these field isolates from Israel could be clustered into all six known clusters. The majority of ARV isolates in Israel belonged to the genotypic cluster 5 (GC5). The strains in this study had a low sequence identity when compared to the commercial vaccine (strain S1133). The findings of this study demonstrated the genetic diversity of ARV strains in Israel from 2015 to 2022. It is reasonable to conclude from the preliminary results of this investigation that Israel has not been subject to selection pressure or the emergence of new ARV variants since the introduction of the live vaccine (ISR-7585). Due to the ongoing emergence of ARV variants, a robust epidemiological monitoring program supported by molecular biology techniques is required to track ARV strains in Israeli poultry flocks.

Psychiatric Comorbidities Associated With Keratoconus.

Importance: Evaluating risk factors for keratoconus, often associated with recurrent eye rubbing, could generate hypotheses to be tested in future interventional trials. Objective: To assess the risk for keratoconus associated with psychiatric comorbidities in adolescents and adults. Design, Setting, and Participants: This population-based cross-sectional study included medical records of Israeli adolescents and adults in military service from January 2011 through December 2021. Main Outcomes and Measures: The prevalence of anxiety, obsessive compulsive disorder (OCD), autism, and attention-deficit/hyperactivity disorder (ADHD) was evaluated in individuals with and without keratoconus. The association between keratoconus and psychiatric comorbidities was tested using univariate and multivariant analyses. Results: Overall, 940 763 adolescents and adults were included. Mean (SD) age was 17.56 (1.47) years, and 59.3% were male. Keratoconus was documented in 1533 individuals, with a prevalence of 0.16%. Patients with keratoconus were more likely to be diagnosed with ADHD compared with the general population (odds ratio [OR], 1.58; 95% CI, 1.38-1.81; P < .001). After adjusting for age, sex, intellectual status, height, and weight, the results remained unchanged (hazard ratio, 1.46; 95% CI, 1.27-1.67; P < .001). Stratification according to age showed an association between keratoconus and ADHD for males (OR, 1.62; 95% CI, 1.39-1.90; P < .001) but not for females (OR, 1.29; 95% CI, 0.96-1.74; P = .09). Conclusions and Relevance: In a large cohort of adolescents and adults, ADHD was associated with a diagnosis of keratoconus in male patients, even after adjusting for possible confounders. Although a causative effect could not be ascribed, these findings support further investigation into the potential value of education regarding eye rubbing in this population.

The association between keratoconus and systemic manifestations of connective tissue hyperlaxity.

PURPOSE: To evaluate the correlation between keratoconus and systemic manifestations of tissue hyperlaxity in the general population. METHODS: In this population based cross-sectional study 940,763 medical records of Israeli adolescents and young adults in military service were reviewed. Demographic and medical data were extracted. The prevalence of ligament injuries, habitual orthopedic deformities and umbilical/inguinal hernia was evaluated in cases with and without keratoconus. The association was tested using uni- and multivariant analyses. RESULTS: Overall 938,411 adolescents and adults were included. Mean age was 17.55 ± 1.50 years, and 40.70% were female. Keratoconus was documented in 1,529 cases, with a prevalence of 0.16%. Compared to the general population, patients with keratoconus were significantly more likely to be diagnosed with genu varum/valgus (OR = 2.75, CI 1.48-5.13, p = 0.0015), pes planus (OR = 1.97, 95% CI: 1.62-2.38, p < 0.0001), scoliosis (OR = 1.88, 95% CI: 1.45-2.43, p < 0.0001) and umbilical/inguinal hernias (OR = 2.18, 95% CI: 1.47-3.24, p = 0.0001). On multivariate analysis the results remained significant (p < 0.05 for all). Joint injuries (ankle sprains, shoulder dislocation and injury to knee ligaments and menisci) were not significantly related to keratoconus (p > 0.05 for all). CONCLUSIONS: In this large cohort of adolescents and young adults, an association was found between keratoconus and connective tissue hyperlaxity manifestations involving the knees, feet, spine and abdomen. These findings suggest that keratoconus might be a manifestation of a generalized connective tissue disorder, rather than just a local ocular phenomenon.

Post-refractive surgery of Israeli Defense Forces recruits in 2005-2018-prevalence, combat unit drop-out rates and utilization of eye-care services.

PURPOSE: To assess the number of recruits for military service in the Israeli Defense Force (IDF) who underwent refractive surgery prior to enlistment and examine whether the procedure affected their ability to accomplish combat training. SETTING: Medical records of IDF recruits. DESIGN: Retrospective analysis of medical records of recruits with ametropia who underwent or did not undergo refractive surgery prior to enlistment. METHODS: Recruits were categorized into ametropes and recruits who underwent refractive surgery. Fitness and assignment to combat units and completion status of combat training were compared between the two groups. RESULTS: The study included 334,688 (182,969 males, 151,719 females) ametropes of which 5231 (4753 males, 478 females) underwent refractive surgery prior to recruitment. Refractive surgery prevalence increased from 9/1000 ametropes in 2005 to 18.5/1000 ametropes in 2018 (r = 0.912, p < 0.001); 2643 of the operated recruits (50.5%) had their surgery at the age of 17-18. Dropout rates from combat training were significantly lower in the refractive surgery group during the study period (1.68% vs. 6.14%, respectively, p < 0.001). Soldiers in the operated group were more frequently referred to ophthalmologists than those in the ametropes group and less frequently referred to optometrists. CONCLUSIONS: The prevalence of refractive surgery in IDF recruits has increased substantially during the last decade with more of them applying to combat units. Refractive surgery opened new possibilities for recruits who were unfit for combat duty prior to surgery and did not appear to impair the chances of successfully completing combat training.

Development of a wide-range real-time RT-PCR assay for detection of Avian reovirus (ARV).

Avian reovirus (ARV) is a common pathogen in chickens and other birds causing a variety of clinical symptoms such as arthritis and tenosynovitis but also enteric and respiratory symptoms. A rapid method that detects as many ARV genotypes as possible, will contribute to the early identification and control of the virus infection that causes high economic damage to the poultry industry worldwide. In this study, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for the detection of ARV was developed. The RT-qPCR detection threshold for ARV genomic RNA standard cases was 10 copies/µL. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assays. When the nucleic acids of different ARV genotypes and other common avian pathogens (IBDV, AIV, NDV, and IBV) were subjected to that RT-qPCR test, only ARV samples tested positive while all other pathogens tested negative. Due to the simplicity, convenience, high sensitivity, and specificity of the assay, the probe-based RT-qPCR is proposed to be used as an alternative diagnostic assay for the detection of ARVs in veterinary diagnostic laboratories.

Tele-ophthalmology as an aid tool for primary care physicians in the IDF, during the Covid-19 lockdown.

PURPOSE: To describe tele-ophthalmology consultations for primary-physicians in the IDF during the Covid-19 lockdown. METHODS: Retrospective cross-sectional study. Medical records of tele-ophthalmology consultations from March to June 2020 were reviewed. RESULTS: The study included 245 cases in which an ophthalmologist was consulted. In 62.0% cell-phone camera photographs were used. The mean age was 21.5 ± 6.4 years. The most common diagnoses were acute-conjunctivitis (8.6%); conjunctival-hyperemia (non-specific diagnosis, 8.2%); scleritis/episcleritis (7.3%); chronic allergic-conjunctivitis (7.3%); chalazion (7.3%) hordeolum (6.5%); acute allergic-conjunctivitis (4.5%). 37.6% of patients received primary physician-based treatment, 24.9% of patients received specialist-based treatment. 13.1% were referred to the ER. The consult prevented ER referral for 39.2% and changed the physician's treatment plan in 70.6% of cases. Foreign-body sensation complaints were more likely treated by a primary-physician (p = 0.015). Cases with suspected foreign-body diagnosis were referred more to the ER (p < 0.001). For most cases of eyelid complaints and diagnoses, primary physician care was sufficient (p < 0.001). Conjunctival complaints and diagnoses received significantly more ophthalmologist-based treatment (p < 0.001). Corneal disorders were significantly referred more to the ER (p = 0.001). CONCLUSION: Despite of possible ethical and legal problems and clinical limitations of this instrument, Tele-ophthalmology using objective aids such as smartphone photography can be an efficient tool in aiding the primary-physician, especially for patients with low access to ophthalmologists, with major impact on patient management.

Physiological response of mammary glands to Escherichia coli infection:A conflict between glucose need for milk production and immune response.

The mammary immune and physiological responses to distinct mammary-pathogenic E. coli (MPEC) strains were studied. One gland in each of ten cows were challenged intra-mammary and milk composition (lactose, fat, total protein, casein), biochemical (glucose, glucose-6-phosphate (Glu6P), oxalate, malate, lactate, pyruvate and citrate, malate and lactate dehydrogenases, lactate dehydrogenase (LDH), nitrite, lactic peroxidase, catalase, albumin, lactoferrin, immunoglobulin) and clotting parameters were followed for 35 days post-challenge. Challenge lead to clinical acute mastitis, with peak bacterial counts in milk at 16-24 h post-challenge. Biochemical and clotting parameters in milk reported were partially in accord with lipopolysaccharide-induced mastitis, but increased Glu6P and LDH activity and prolonged lactate dehydrogenase and Glu6P/Glu alterations were found. Some alterations measured in milk resolved within days after challenge, while others endured for above one month, regardless of bacterial clearance, and some reflected physiological responses to mastitis such as the balance between aerobic and anaerobic metabolism (citrate to lactate ratios). The results suggest that E. coli mastitis can be divided into two stages: an acute, clinical phase, as an immediate response to bacterial infection in the mammary gland, and a chronic phase, independent of bacteria clearance, in response to tissue damage caused during the acute phase.

Reversal of cystoid macular edema in gyrate atrophy patients.

PURPOSE: This study reports the presentation of two families with gyrate atrophy (GA). The aim of this study was to characterize the potential effect of therapeutic regimens on macular edema. METHODS: Two unrelated patients with GA were studied for the potential effect of low protein diet (≤ 0.8 g/kg/d), and oral administration of pyridoxine (500 mg/day), on serum ornithine levels, best corrected visual acuity (BCVA), slit-lamp, OCT, and auto-fluorescence findings. Blood samples for DNA, mRNA, and exons of the OAT gene were screened for mutations and splicing effect when relevant. RESULTS: At presentation, both patients manifested typical ophthalmic features of GA including cystoid macular edema (CME). One patient also exhibited optic nerve head hamartoma. Following treatment ornithine levels have lessened, BCVA improved, and central macular thickness (CMT) markedly decreased in all four studied eyes. The molecular pathologic features included a novel splice site mutation (c.900+1G>A). CONCLUSIONS: We have identified a novel mutation and two formerly described mutations in patients with GA. Of them, one patient comprised an unusual phenotype including bilateral astrocytic hamartomas. We have recognized for the first time improvement in CME following treatment with low protein intake and pyridoxine supplement. This finding may have significance in the understanding of treatment options for macular edema regardless of underlying etiology.

Digoxin derivatives with selectivity for the α2ß3 isoform of Na,K-ATPase potently reduce intraocular pressure.

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1ß1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2ß3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2ß3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2ß1 over α1ß1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the ß1 subunit, Gln84, suggesting a role for ß in digoxin binding. Gln84 in ß1 is replaced by Val88 in ß3. Assuming that alkyl substituents might interact with ß3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2ß3 over α1ß1 (22-33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2ß3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1ß1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2ß3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1ß1, α2ß1, and α3ß1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH2 substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.

The host genotype influences infectious bursal disease virus pathogenesis in chickens by modulation of T cells responses and cytokine gene expression.

The immunopathogenesis of infectious bursal disease virus (IBDV) was investigated in different layer and broiler type chickens in comparison to highly susceptible specific-pathogen-free layers (SPF-Wh-LT) often used for experimental studies. Layer-type chickens (LT) of all genetic backgrounds showed significantly higher IBDV antigen loads in the bursa of Fabricius (BF) compared to broiler type birds (BT) (P<0.05). The variation between IBDV-infected and virus-free birds in the percentage of splenic and intrabursal B cells, T cells and macrophages differed between genetic backgrounds as well as the expression levels of cytokines. The most susceptible SPF-Wh-LT showed high levels of circulating type I IFN starting at 2 days post infection (dpi) up to 7 dpi coinciding with clinical IBD, while less susceptible birds showed a delayed response. Circulating cytokine levels were poorly associated neither with intrabursal nor with splenic mRNA expression of these cytokines. Detected cytokines varied in expression levels and timing between infected groups of different genetic background. These data suggest that variations in the activity of immune cell populations contribute to differences in infectious bursal disease between birds of various genetic backgrounds.

Contribution of the genetic background to the immune response of broilers vaccinated or challenged with LPAI H9N2.

BACKGROUND: The knowledge on the immune responses to LPAI is limited. The purpose of this study was to investigate the immune responses of two divergently selected lines of broilers, a line responding with high antibody response to antigens (HH), and a line responding with low antibody titers (LL) to antigen. METHODS: Day old chicks from each line were divided in two groups, one vaccinated with inactivated H9N2 vaccine and one non-vaccinated. At 21 days of age all the chicks were challenged with field isolate of H9N2, 1X106.5 ELD50 per chick by drops to the eye, nose and beak. Twenty four hours and 14 days post challenge (PC), the chickens were weighed blood spleen and lungs were taken and leukocytes were isolated. The leukocytes were stained with monoclonal antibodies for surface markers and analyzed by flow cytometry. We used Elispot assay to identify the number of antibody producing cells in each of the organs. mRNA was extracted using TRIsol reagent in order to assess the cytokine production level by qRT-PCR using the SYBR green methods. RESULTS: Our results showed that LL-vaccinated group gained more weight than any of the other group. Using IDEXX kit, no antibody titers could be identified in vaccinated chicks 21 days post vaccination while 14 days PC vaccinated HH chickens demonstrated the highest average antibody titers. LL vaccinated chickens demonstrated higher average antibody titer than non-vaccinated LL. Using the Elispot assay no difference were found between the groups either cells producing IgA, IgM or IgY beside of a high number of IgY producing cells in the lungs of vaccinated HH birds. CONCLUSIONS: Further data on leukocytes subpopulations using flow cytometry, cytokines production (IFNγ, IL-6, IL-18, IL-2 and IL-4) isotype specific antibody responses and number and functionality of NK cells are in process.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.