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Background and Aim: Rabbit hemorrhagic disease (RHD) is an economically important disorder of rabbits, where infection results in severe losses to the meat and fur industries. Our goal was to characterize the RHD virus (RHDV) strains currently circulating in different regions of Egypt. Materials and Methods: Fifty rabbits suspected of harboring RHDV from 15 Egyptian governorates were evaluated. Diseased rabbits were identified by clinical signs and postmortem lesions. RHDV was confirmed through hemagglutination assay (HA) and polymerase chain reaction (PCR). Partial sequencing of the VP60 gene was performed for genotyping. Results: From 50 rabbits, we identified 16 cases of RHDV (32%) by HA and PCR, including seven males and nine females. We identified two distinct genotypes through sequencing of an amplified fragment of the virus VP 60 gene. One group is composed of those circulating primarily in upper Egypt, which is closely related to the classical G3-G5 virus strains, and the second group, circulating predominantly in lower Egypt, was more closely related to the RHDV2 variant. The overall nucleotide sequence identity ranged from 78.4% to 100%, and identity with the vaccine strains ranged from 78.8% to 91.1%. Conclusion: Our results constitute important documentation of RHDV strains currently circulating in Egypt. The findings suggest that there may be a limit to the effectiveness of currently applied vaccine strains as this formulation may not cover all circulating strains. A wider investigation that includes both domestic and wild rabbits will be needed to identify appropriate control measures for this disease.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019–2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019–2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020–2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

In Egypt, ducks kept for commercial purposes constitute the second highest poultry population, at 150 million ducks/year. Hence, ducks play an important role in the introduction and transmission of avian influenza (AI) in the Egyptian poultry population. Attempts to control outbreaks include the use of vaccines, which have varying levels of efficacy and failure. To date, the effects of vaccine efficacy has rarely been determined in ducks. In this study, we evaluated the protective efficacy of a live recombinant vector vaccine based on a turkey Herpes Virus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAIV strain (A/Swan/ Hungary/499/2006) (rHVT-H5) and a bivalent inactivated H5N1 vaccine prepared from clade 2.2.1 and 2.2.1.1 H5N1 seeds in Mulard ducks. A 0.3ml/dose subcutaneous injection of rHVT-H5 vaccine was administered to one-day-old ducklings (D1) and another 0.5ml/ dose subcutaneous injection of the inactivated MEFLUVAC was administered at 7 days (D7). Four separate challenge experiments were conducted at Days 21, 28, 35 and 42, in which all the vaccinated ducks were challenged with 106EID50/duck of H5N1 HPAI virus (A/ chicken/Egypt/128s/2012(H5N1) (clade 2.2.1) via intranasal inoculation. Maternal-derived antibody regression and post-vaccination antibody immune responses were monitored weekly. Ducks vaccinated at 21, 28, 35 and 42 days with the rHVT-H5 and MEFLUVAC vaccines were protected against mortality (80%, 80%, 90% and 90%) and (50%, 70%, 80% and 90%) respectively, against challenges with the H5N1 HPAI virus. The amount of viral shedding and shedding rates were lower in the rHVT-H5 vaccine groups than in the MEFLUVAC groups only in the first two challenge experiments. However, the non-vaccinated groups shed significantly more of the virus than the vaccinated groups. Both rHVT-H5 and MEFLUVAC provide early protection, and rHVT-H5 vaccine in particular provides protection against HPAI challenge.

Background and Aim: Rabbits are a highly sensitive species and susceptible to various bacterial pathogens that may be causative agents for early embryonic death. This study aimed to explore the administration of different bacterial agents in does suffering from early embryonic death. Furthermore, identification of genes associated with virulence was performed to identify the phenotypic and genotypic antimicrobial resistance patterns that may increase the virulence of pathogens and lead to early embryonic death. Materials and Methods: We isolated and identified bacterial agents in 106 samples from live and dead female rabbits that had undergone early embryonic death, including liver and intestine tissue, aborted fetuses, discharges, and vaginal swabs. Conventional polymerase chain reaction (PCR) was conducted to confirm the identity of the isolated bacterial strains and their virulence. Moreover, antibiotic resistance was studied phenotypically and genotypically. Results: We isolated Escherichia coli, Salmonella, Staphylococcus aureus, Pasteurella multocida, and Listeria monocytogenes. PCR confirmed typical identification except in P. multocida, which was confirmed as Gallibacterium spp. in some cases. The final percentage of detection was 34%, 30.2%, 16.9%, 13.2%, and 11.3%, respectively. Virulence properties were investigated using different designated genes. All Salmonella strains harbored invA, stn, avrA, and ompf genes, while the sopE gene was identified in 31.25%. E. coli strains harboring the iss gene lacked the shiga toxin (stx1) gene. L. monocytogenes and S. aureus strains harbored the hemolysin gene (66.7% and 33.4%, respectively). Multidrug resistance was detected phenotypically and genotypically in most strains. Each bacterial pathogen had a different antibiotic resistance profile. Conclusion: Multiple bacterial species may contribute to early embryonic death in does. Furthermore, the combined infection could be the main cause of early embryonic death. Thus, monitoring programs should bear this in mind and focus on the early detection of these bacterial agents in female rabbits to avoid embryonic death.

Background and Aim: Flinders Technology Associates (FTA) cards simplify sample storage, transport, and extraction by reducing cost and time for diagnosis. This study evaluated the FTA suitability for safe transport and storage of Gram-positive and Gram-negative bacterial cells of animal origin on its liquid culture form and from organ impression smears (tissues) under the same routine condition of microbiological laboratory along with detecting their nucleic acid over different storage conditions. Materials and Methods: Increase in bacterial count from 104 to 107 (colony-forming units/mL) of 78 isolates representing seven bacterial species was applied onto cards. FTA cards were grouped and inoculated by these bacteria and then stored at different conditions of 24-27°C, 4°C, and –20°C for 24 h, for 2 weeks, for 1 and 3 month storage, respectively. Bacteriological examination was done, after which bacterial DNA was identified using specific primers for each bacterial type and detected by polymerase chain reaction (PCR). Results: The total percentage of recovered bacteria from FTA cards was 66.7% at 24-27–C for 24 h, the detection limit was 100% in Gram-positive species, while it was 57.4% in Gram-negative ones. Regarding viable cell detection from organ impression smears, it was successful under the previous conditions. No live bacterial cells were observed by bacteriological isolation rather than only at 24-27°C for 24 h storage. All bacterial DNA were sufficiently confirmed by the PCR technique at different conditions. Conclusion: Overall, the FTA card method was observed to be a valid tool for nucleic acid purification for bacteria of animal origin in the form of culture or organ smears regardless of its Gram type and is used for a short time only 24 h for storage and transport of live bacteria specifically Gram-positive type. Moreover, the bacterial nucleic acid was intact after storage in –20°C for 3 months and was PCR amplifiable.

A total of 220 birds of age ranging from 3 to 14 weeks old were collected from several backyards and different farms in Sharkia Governorate, Egypt, and surveyed for the presence of fowl cholera. Twenty Pasteurella multocida from chickens (15/145, 10%) and ducks (5/75, 6%) were bacteriologically isolated, and it was shown that the infection was significantly related to age and breed. Capsular typing, using multiplex polymerase chain reaction (PCR), demonstrated that all strains were type A (100%). Disk diffusion assay towards ten antimicrobials revealed high susceptibilities to amikacin, doxycycline, chloramphenicol, and neomycin with varying degrees. Doxycycline was effective at the lowest concentration (MIC 0.125–1 µg/ml). Multidrug resistance was detected with a percentage of 25%. Multidrug-resistant isolates (five isolates) were subjected to study their pathogenicity in embryonated chicken eggs (ECE). The results showed a variation in indices between different dilutions of the tested strains. The resulting pathogenicity indices showed significant differences (P < 0.05) according to the origin and dilution of the isolate. From the original inoculum to 10–4 dilutions, the mortality of inoculated embryos occurred within 1–2 days with pathological findings, including maceration and lesions on chorioallantoic membrane (CAM). From dilutions ranging from 10–5 to 10–9, no death occurred until 7 days post-inoculation, but a variation in the lesions on CAM was observed. In conclusion, P. multocida serogroup A could be intensely pathogenic for mature chickens thus causing considerable economic losses, and PCR provides a suitable technique for early and rapid diagnosis of fowl cholera.

Background and Aim: Rabbit hemorrhagic disease (RHD) is an economically important disorder of rabbits, where infection results in severe losses to the meat and fur industries. Our goal is to characterize the RHD virus (RHDV) strains currently circulating in different regions of Egypt. Materials and Methods: Fifty rabbits suspected of harboring RHDV from 15 Egyptian governorates were evaluated. Diseased rabbits were identified by clinical signs and postmortem lesions. RHDV was confirmed through hemagglutination assay (HA) and polymerase chain reaction (PCR). Partial sequencing of the VP 60 gene was performed for genotyping. Results: From 50 rabbits, we identified 16 cases of RHDV (32%) by HA and PCR, including seven males and nine females. We identified two distinct genotypes through sequencing of an amplified fragment of the virus VP 60 gene. One group is composed of those circulating primarily in upper Egypt, which is closely related to the classical G3-G5 virus strains, and the second group, circulating predominantly in lower Egypt, was more closely related to the RHDV2 variant. The overall nucleotide sequence identity ranged from 78.4% to 100%, and identity with the vaccine strains ranged from 78.8% to 91.1%. Conclusion: Our results constitute important documentation of RHDV strains currently circulating in Egypt. The findings suggest that there may be a limit to the effectiveness of currently applied vaccine strains as this formulation may not cover all circulating strains. A wider investigation that includes both domestic and wild rabbits will be needed to identify appropriate control measures for this disease. Keywords: Egypt, native rabbits, prevalence, rabbit hemorrhagic disease virus, VP 60.