Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt.

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

Serological Investigation on the Presence of Feline Coronavirus (FCoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Domestic Cats Living with COVID-19 Positive Owners in the UAE, 2022.

Feline coronavirus (FCoV) is widely circulating among domestic cats (Felis catus). The zoonotic origin of the emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the biological characteristics of CoVs, including the ability to cross interspecies barriers, facilitate its emergence in different animals, including cats' populations. The current study is the first to report the serological investigation on the presence of FCoV and SARS-CoV-2 in domestic cats living with COVID-19-positive owners in the UAE. A total of 83 sera were collected from domestic cats living with COVID-19-positive owners (by RT-qPCR). The cats were sampled during the period between February and May 2022 in Al-Ain and Abu Dhabi Cities, UAE. Detection of FCoV and SARS-CoV-2 was carried out by enzyme-linked immunosorbent assay (ELISA). FCoV antibodies were detected in 54 samples (65%). The frequencies of FCoV were significantly higher in purebred cats (48%; 40/83) and in the cat group with outdoor access (49.4%; 41/83). SARS-CoV-2 seroprevalence in collected sera revealed 8 samples (9.6%) with positive results. Four samples (4.8%) showed positive results for both FCoV and SARS-CoV-2 antibodies. In conclusion, FCoV is widely circulating within cats' populations involved in the study. The antibodies for SARS-CoV-2 were detected in cats' populations but at a low prevalence rate. COVID-19-positive people should avoid close contact with their cats. Future serological testing of large cats' populations is crucial for providing a good understanding of COVID-19 dynamics in cats.

Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt.

In the summers of 2018 and 2019, a disease outbreak stroke 25 broiler chicken farms and 3 broiler breeder farms in different Governorates in Egypt. The disease caused a mortality rate ranging from 3.2 to 9%. Postmortem examination showed petechial hemorrhage in the breast and thigh muscles, thymus gland, and peritoneal cavity and extensive hemorrhages in the kidneys. A total of 140 liver, kidney, lung, skeletal muscles, thymus, and spleen samples were collected. Twenty-eight pooled samples were created and examined by PCR and histopathological examination to identify the causative pathogens. All collected samples were PCR-negative to Newcastle disease virus (NDV), avian influenza viruses (H5, H9, and H7), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and fowl adenovirus (FadV). Leucocytozoon caulleryi (L. caulleryi) genetic material was identified by PCR in 17 out of the 28 collected samples (61%). Five chicken farms (18%) showed positive PCR results for both L. caulleryi and chicken anemia virus (CAV). Histopathological examination revealed unilocular megaloschizonts in thymus, skeletal muscle, and lung as well as massive hemorrhages in parenchymatous organs. Nucleotide sequences of the identified pathogens were compared with other reference sequences available in the GenBank. The identified L. caulleryi has a close relationship with those previously detected in Asia, indicating potential transmission route of the parasite. The CAV has a close genetic relation with CAVs previously identified in Egypt. Furthermore, a real-time PCR for rapid, specific, and quasiquantitative detection of L. caulleryi was developed with a detection limit of 100 genome copies per reaction.

Green synthesized silver nanoparticles using Cyperus rotundus L. extract as a potential antiviral agent against infectious laryngotracheitis and infectious bronchitis viruses in chickens.

Background: Infectious laryngotracheitis (ILT) and infectious bronchitis (IB) are two common respiratory diseases of poultry that inflict great economic burden on the poultry industry. Developing an effective agent against both viruses is a crucial step to decrease the economic losses. Therefore, for the first time green synthesized silver nanoparticles using Cyperus rotundus L. aqueous extract was evaluated in vitro as a potential antiviral against both viruses. Results: Silver nanoparticles from Cyperus rotundus were characterized by the spherical shape, 11-19 nm size, and zeta potential of - 6.04 mV. The maximum nontoxic concentration (MNTC) was 50 µg mL-1 for both viruses without harmful toxicity impact. The study suggested that some of the compounds in C. rotundus extract (gallic acid, chlorogenic acid, and naringenin) or its silver nanoparticles could interact with the external envelope proteins of both viruses, and inhibiting extracellular viruses. Conclusions: The results highlight that C. rotundus green synthesized silver nanoparticles could have antiviral activity against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) in chickens.

Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip.

Infectious laryngotracheitis (ILT) is a viral disease of chickens' respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases.

Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

The detection dogs test is more sensitive than real-time PCR in screening for SARS-CoV-2.

In January 2020, the coronavirus disease was declared, by the World Health Organization as a global public health emergency. Recommendations from the WHO COVID Emergency Committee continue to support strengthening COVID surveillance systems, including timely access to effective diagnostics. Questions were raised about the validity of considering the RT-PCR as the gold standard in COVID-19 diagnosis. It has been suggested that a variety of methods should be used to evaluate advocated tests. Dogs had been successfully trained and employed to detect diseases in humans. Here we show that upon training explosives detection dogs on sniffing COVID-19 odor in patients' sweat, those dogs were able to successfully screen out 3249 individuals who tested negative for the SARS-CoV-2, from a cohort of 3290 individuals. Additionally, using Bayesian analysis, the sensitivity of the K9 test was found to be superior to the RT-PCR test performed on nasal swabs from a cohort of 3134 persons. Given its high sensitivity, short turn-around-time, low cost, less invasiveness, and ease of application, the detection dogs test lends itself as a better alternative to the RT-PCR in screening for SARS-CoV-2 in asymptomatic individuals.

A portable, 3D printed, microfluidic device for multiplexed, real time, molecular detection of the porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine deltacoronavirus at the point of need.

The porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are emerging/reemerging coronaviruses (CoVs) of neonatal pigs that cause great economic losses to pig farms and pork processors. Specific, rapid, and simple multiplex detection of these viruses is critical to enable prompt implementation of appropriate control measures. Conventional methods for molecular diagnosis require skilled personnel and relatively sophisticated equipment, restricting their use in centralized laboratories. We developed a low-cost, rapid, semi-quantitative, field deployable, 3D-printed microfluidic device for auto-distribution of samples and self-sealing and real-time and reverse transcription-loop-mediated isothermal amplification (RT-LAMP), enabling the co-detection of PEDV, TGEV and PDCoV within 30 minutes. Our assay's analytical performance is comparable with a benchtop, real-time RT-LAMP assay and the gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay with limits of detection of 10 genomic copies per reaction for PEDV and PDCoV, and 100 genomic copies per reaction for TGEV. Evaluation of clinical specimens from diseased pigs with our microfluidic device revealed excellent concordance with both benchtop RT-LAMP and qRT-PCR. Our portable RT-LAMP microfluidic chip will potentially facilitate simple, specific, rapid multiplexed detection of harmful infections in minimally equipped veterinary diagnostic laboratories and on-site in pigs' farms.

Two stage, nested isothermal amplification in a single tube.

Sensitive, specific and rapid molecular diagnosis of respiratory diseases in animals and humans is critical to facilitate appropriate control measures and treatment. Conventional polymerase chain reaction (PCR)-based molecular diagnostics requires relatively expensive equipment and trained staff, restricting its use to centralized laboratories with significant delays between sample collection and test results. Herein, we report a highly sensitive, rapid, point-of-need, two-stage-molecular test that requires minimal instrumentation and training. Our test, dubbed Penn-RAMP, combines recombinase polymerase amplification (RPA, 38 °C) and loop-mediated isothermal amplification (LAMP, 63 °C) in one tube, enabling nested, two-stage isothermal amplification. We demonstrate Penn-RAMP's efficacy by testing for two common viral respiratory diseases of chickens: infectious laryngotracheitis (ILT) and infectious bronchitis (IB) that impose great economic burden worldwide. Test results of clinical samples with our closed-tube Penn-RAMP assays concord with the gold standard quantitative PCR (qPCR) assay; with 10-fold better limit of detection than LAMP and qPCR. Our closed-tube Penn-RAMP assays have the potential to greatly reduce false negatives while requiring minimal instrumentation and training.

Molecular investigation on the presence of canine parvovirus in Egypt.

Canine parvovirus type 2 (CPV-2) causes a highly contagious gastroenteritis disease of dogs and wild canids. To investigate the CPV-2 prevalence in Dakahlia Governorate, Egypt, a total of 50 fecal swabs were collected from suspected diseased dogs during 2016-2017. Out of 50 collected samples, 35 samples (70 %) presented positive results for CPV-2 using immuno-chromatography (IC) as a rapid test. CPV-2DNA was detected in 42 samples (84 %) by using polymerase chain reaction (PCR). The frequencies of CPV-2 were significantly higher in German shepherd breed (46 %; 23/50) and in age groups less than 6 months (76%; 38/50). We evaluated the breed, age, sex, rapid test results and clinical signs as predictors for classification of animal status into infected and not infected. The best predictors for classification process were rapid test result and clinical signs. Both CPV-2b and CPV-2c subtypes were detected by CPV2-VP2 gene sequences analysis. Deduced amino acid sequences alignment showed substitutions at 3 sites (Arg453Pro, Ala574Glu and Gln457Leu). Further investigations are needed to reveal the genetic and antigenic relation between field and vaccinal strains of CPV-2 in Egypt.

Molecular Characterization and Developing a Point-of-Need Molecular Test for Diagnosis of Bovine Papillomavirus (BPV) Type 1 in Cattle from Egypt.

Bovine papillomatosis is a viral disease of cattle causing cutaneous warts. A diagnosis of this viral infection is very mandatory for combating the resulting economic losses. Given the limited data available about bovine papillomavirus (BPV) in Egypt, the present study involved the molecular diagnosis of bovine papillomavirus type-1 (BPV-1), -2, -4, -5, and -10 in cattle presenting cutaneous warts on the head and neck from New Valley Province, Egypt. The phylogenetic analysis of the detected types of BPV was also performed, followed by developing a point-of-need molecular assay for the rapid identification of identified BPV types. In this regard, a total of 308 cattle from private farms in Egypt were clinically examined, of which 13 animals presented cutaneous warts due to suspected BPV infection. The symptomatic animals were treated surgically, and biopsies from skin lesions were collected for BPV-1, -2, -4, -5, and -10 molecular identification using polymerase chain reaction (PCR). The presence of BPV-1 DNA was confirmed in 11 collected samples (84.6%), while BPV-2, -4, -5, and -10 were not detected. Sequencing of the PCR products suggested the Egyptian virus is closely related to BPV found in India. An isothermal nucleic acid amplification test (NAAT) with labeled primers specific for the BPV-1 L1 gene sequence, and based on recombinase polymerase amplification (RPA), in combination with a lateral flow strip assay for the detection of RPA products, was developed and tested. The point-of-need molecular assay demonstrated a diagnostic utility comparable to PCR-based testing. Taken together, the present study provides interesting molecular data related to the occurrence of BPV-1 in Egypt and reveals the genetic relatedness of the Egyptian BPV-1 with BPV-1 found in buffalo in India. In addition, a simple, low-cost combined test was also validated for diagnosis of the infection. The present study suggests the necessity of future investigations about the circulating strains of the virus among the cattle in Egypt to assess their genetic relatedness and better understand the epidemiological pattern of the disease.

Molecular characterization and pathogenicity of very virulent infectious bursal disease virus isolated from naturally infected turkey poults in Egypt.

This study was conducted to investigate the molecular characterization and pathogenicity of very virulent infectious bursal disease virus (vvIBDV) isolated from naturally infected turkey poults and possible spread to chickens. Thirty samples were collected from turkey poults in the vicinity or in the same backyards with chickens suspected to be infected with IBDV and from live bird markets from different localities in Dakahlia governorate, Egypt. There were no obvious clinical signs in tested turkey poults except dehydration and whitish diarrhoea in some birds with no mortality, and post-mortem lesions were observed in few birds as atrophied bursae, nephritis and petechial haemorrhages on thigh muscles. Reverse transcription polymerase chain reaction (RT-PCR), histopathological examination and immunohistochemistry were used for identification of the IBDV. Out of 30 tested samples, 17 samples (56.7%) were positive by RT-PCR. Phylogenetic analysis of VP2 gene of two selected IBDV strains (turkey 1 and turkey 2) showed a close genetic relationship to vvIBDV strains (serotype 1) isolated from chickens in Egypt and other countries with 93.1 to 95.99% identity for turkey 1 strain and 95.54 to 98.51% for turkey 2 strain. Both turkey 1 and turkey 2 strains were closely related to the Nigerian vvIBDV strain isolated from turkeys with 95.78% and 96.37% identity, respectively. Sequence analysis of both strains demonstrated that they have conserved amino acid residues of vvIBDV (I242, I294 and S299) and Y220F amino acid substitution which is very common in Egyptian vvIBDV chicken strains, while Turkey 1 strain has amino acid substitutions at A222P and I256V. Histopathological examination showed marked depletion of bursal lymphoid tissue. In conclusion, for the first time in Egypt, the molecular characterization and pathogenicity confirmed the presence of natural infection of turkey poults with vvIBDV (serotype 1) with possible spread to chickens causing severe economic losses.

Molecular Detection of Reticuloendotheliosis Virus 5' Long Terminal Repeat Integration in the Genome of Avipoxvirus Field Strains from Different Avian Species in Egypt.

Avipoxviruses (APVs) are among the most complex viruses that infect a wide range of birds' species. The infection by APVs is often associated with breathing and swallowing difficulties, reduced growth, decreased egg production, and high mortalities in domestic poultry. In the present study, 200 cutaneous nodular samples were collected from different avian species (chicken, pigeon, turkey, and canary) suspected to be infected with APVs from Dakahlia Governorate, Egypt. Pooled samples (n = 40) were prepared and inoculated in embryonated chicken eggs (ECEs). APVs were then identified by polymerase chain reaction (PCR) and sequence analysis of the APV P4b gene. Furthermore, the forty strains of APVs were screened for the presence of reticuloendotheliosis virus (REV)-5'LTR in their genomes. Interestingly, the phylogenic tree of the APV P4b gene was separated into 2 clades: clade 1, in which our fowlpox virus (FWPV), turkeypox virus (TKPV), and canarypox virus (CNPV) isolates were grouped, along with reference FWPVs and TKPVs retrieved from GenBank, whereas, in clade2, the pigeonpox virus (PGPV) isolate was grouped with PGPVs retrieved from GenBank. Likewise, REV-5'LTR was amplified from 30 strains isolated from chicken, turkey, and canary, while PGPV strains were free from REV-5'LTR integration. To the best of our knowledge, this study involved the detection and characterization of REV-5'LTR insertions in the APVs field isolates in Egypt for the first time. Given the above information, further future research seems recommended to understand the impact of the resulting REV-5'LTR insertions on the pathogenesis, virulence, and inadequate vaccine protection against APVs.

A closed-tube, single-step, real time, reverse transcription-loop-mediated isothermal amplification assay for infectious bronchitis virus detection in chickens.

Infectious bronchitis (IB) is a viral infection of the chicken respiratory tract that causes substantial economic burden on the industry. Simple, specific and rapid diagnosis of this disease is critical for the initiation of appropriate control measures. Conventional molecular diagnostic methods require a relatively sophisticated equipment and skilled staff. Here we describe a rapid, simple, semi-quantative, closed-tube, single-step, real-time- reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for IB and compare our assay with quantative, reverse transcription- polymerase chain reaction (RT-qPCR). The limit of detection (LOD) of our RT-LAMP assay is 1 EID50/ ml. Clinical evaluation of samples from diseased chickens with our RT-LAMP showed a very good concordance with RT-qPCR. Our assay enables simple, specific, rapid molecular detection and semi-quantification of the infectious bronchitis virus (IBV) in veterinary diagnostic laboratories. Furthermore, our RT-LAMP detection is carried out in a sealed tube, eliminating the risk of false-positive results in subsequent tests because of any contamination of the work area as in the case of lateral flow strip or gel electrophoresis-based amplicon detection.

A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry.

The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training.

Dataset of the microbiome composition in skin lesions caused by lumpy skin disease virus via 16s rRNA massive parallel sequencing.

Lumpy Skin Disease (LSD) is a highly contagious viral disease affecting cattle mainly and induced by the Lumpy Skin Virus within the Capripoxvirus genus of the family Poxviridae. LSD infected animals exhibit pyrexia and sudden appearance of localized or generalized skin nodules that may slough leaving ulcers. The disease has negative economic impacts as a result of hide damage, mastitis, infertility and losses in milk production. Secondary bacterial infection in the affected skin lesions can increase the severity and prolong the course of the disease. Little is known about the microbiome in the ulcerated skin sites. Therefore, the present study was directed to identify the prevalent bacterial communities in affected lesion via the 16s rRNA gene sequencing. Up to 98 species were found in the samples, most of them belonging to the phyla of Proteobacteria, followed by Firmicutes, Actinobacteria, and Bacteroidetes. All found bacterial species are known as opportunistic pathogens, but can withstand the inflammatory reaction.

Recombinase polymerase amplification-nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection.

Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)-nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.

Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis from broiler chickens in Egypt.

A case-control study was performed to assess prescence of inclusion body hepatitis (IBH) caused by fowl adenoviruses (FAdVs) at Kafr EL-Shiekh Governorate, Egypt, during spring, 2017. The case group consisted of 100 liver and spleen samples collected from 10 broiler chickens flocks (10 samples from each flock) suspected to be infected with IBH depending on clinical manefestations and necropsy examination. Controls were randamly selected from chickens without clinical sings or evidence of the disease on postmortem examination. Molecular screening of the disease disease in collected samples based on the DNA polymerase gene of FAdVs was carried out. Furthermore, the DNA polymerase gene sequence was determined and analyzed with published reference sequences on GeneBank. Respectively, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm existence of co-infection with chicken infectious anemia virus (CIAV) and/or infectious bursal disease virus (IBDV in flocks involved in the study. Using PCR, FAdV genome was detected in seven flocks in the case group and one in the control group. FAdV identified in this study revealed close genetic relationship with FAdVs-D previously identified in UK and Canada, suggesting potential virus transmission from these countries. All tested serum samples from diseased chickens were positive for CIAV infection via ELISA while none of the collected bursa of Fabricius samples tested IBDV positive by RT-PCR. Therefore, results obtained from the current study highlighted the importance of implementation of control measures against FAdV and CIAV in Egyptian poultry flocks. This study opens the door for future work toward specific identification of FAdV serotypes circulating in Egyptian poultry farms and molecular characterization of the virus based on hexon gene or full genome sequencing for better understanding of genetic diversity among FAdVs in Egypt at higher reolution.

Molecular and pathological characterization of duck enteritis virus in Egypt.

In winter 2016, a fatal disease outbreak suspected to be duck virus enteritis (DVE) stroke over a million ducklings in 10 white Pekin and Muscovy ducks flocks in Dakahlia and Gharbia Governorates, Egypt, causing heavy economic losses. The disease quickly killed 20%-60% of affected farms. The clinical signs were inappetence, ataxia, crowding in corners, partially closed eye lids and blue beaks. Post mortem examination revealed white necrotic foci in liver, mottled spleen and sometimes cecal core. A total of 10 intestines, livers and spleens samples were collected from diseased flocks. Each sample was pooled randomly from eight to ten ducklings. Polymerase chain reaction (PCR) and histopathological examination were utilized for DEV identification in collected samples. Nucleotides sequences of the amplified DNA polymerase gene were compared with the other DEVs available on GeneBank. Also, existence of co-infection with Salmonella spp. was verified via PCR. DEV nucleic acid was detected by PCR in 8 of 10 collected samples (80%) with positive amplification of polymerase gene. Histopathological examination revealed eosinophilic and basophilic intranuclear inclusion bodies in enterocytes. In some infected enterocytes, intranuclear and intracytoplasmic inclusions were observed in the same cell. Respectively, eosinophilic intranuclear inclusion bodies found in hepatocytes and reticular cells of liver and spleen of diseased ducklings. Four of the 10 collected samples showed positive results for Salmonella spp. infection that may be involved in enhancing infection with DEV. The identified DEVs revealed close genetic relationship with DEVs detected previously in India and China indicating potential transmission of the virus from there that crucially needs further work for better understanding of virus origin. In conclusion, our study revealed infection of duckling farms with DEV and Salmonella that necessitate the implementation of restricted early preventive and control measures for both diseases to decrease the expected economic losses.

Implementation of Active Learning Approach to Teach Biorisk Management and Dual-Use Research of Concern in Egypt.

Introduction: Frequent reports of laboratory- and hospital-acquired infection in Egypt suggested a deficiency in handling hazardous samples and microorganisms among different researchers and professionals. The most common cause of laboratory incidents and potential exposure is often identified as a lack of biosafety training. Methods: In this study, we designed and implemented an effective laboratory biorisk management (BRM) training. Two workshops were delivered to 42 faculty members working in laboratories handling biological material in Egypt. The workshop modules were based on the global biorisk management curriculum developed by Sandia National Laboratories, with some modifications. The content was delivered to actively engaging participants in the learning process that included group work, case studies and scenarios, short presentations, demonstrations, hands-on activities, and questions and answers that created analytical thinking situations. These workshops introduced the concept of biorisk management, which combines risk assessment, risk mitigation, and performance systems and dual-use research of concern. Results: Results of pre-tests/post-tests revealed significant (P < .001) improvement in knowledge acquisition among participants. Course evaluation surveys indicate that most participants felt that these teaching methods met their needs and that their personal laboratory practices would change as a result of the training course. Conclusion: We conclude that using varied hands-on strategies in teaching biorisk management provided the participants with the skills, tools, and confidence to guide their laboratory staff and colleagues on sustainable biorisk management to reduce the risks associated with infectious disease research in a laboratory setting.