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.

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.

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 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 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.