Protective efficacy of recombinant turkey herpes virus (rHVT-H5) and inactivated H5N1 vaccines in commercial Mulard ducks against the highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1 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.S1 Table. Weekly mean HI titres (log2 ± SD) using A/Swan/Hungary/4999/2006) rHVT/Ag that indicate the immune response to the rHVT-H5 vaccination. S1 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group I (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).S2 Table. Weekly mean HI titres (log2 ± SD) measured using (A/chicken/Egypt/Q1995D/ 2010) V/H5N1/Ag that indicates the immune response to the KV-H5 vaccination. S2 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group 1 (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).S3 Table. Weekly mean HI titres (log2 ± SD) measured using (A/chicken/Egypt/128S/2012) C/H5N1/Ag that indicates the immune response to the challenge virus. S3 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group 1 (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).This work was supported by the United States Agency for International Development (USAID) under a grant (AID-263-IO-11-00001, Mod. #3) and within the framework of OSRO/EGY/101/ USA, which applies to projects jointly implemented by the FAO, GOVS and NLQP.http://www.plosone.orgam2016Production Animal StudiesVeterinary Tropical Disease

Protective efficacy of recombinant turkey herpes virus (rHVT-H5) and inactivated H5N1 vaccines in commercial Mulard ducks against the highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1 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.S1 Table. Weekly mean HI titres (log2 ± SD) using A/Swan/Hungary/4999/2006) rHVT/Ag that indicate the immune response to the rHVT-H5 vaccination. S1 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group I (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).S2 Table. Weekly mean HI titres (log2 ± SD) measured using (A/chicken/Egypt/Q1995D/ 2010) V/H5N1/Ag that indicates the immune response to the KV-H5 vaccination. S2 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group 1 (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).S3 Table. Weekly mean HI titres (log2 ± SD) measured using (A/chicken/Egypt/128S/2012) C/H5N1/Ag that indicates the immune response to the challenge virus. S3 Table legend: Different upper case letters in a row denote the presence of statistically significant (p 0.05) differences. Group 1 (vaccinated with rHVT-H5 vaccine at 1 day old), Group II (vaccinated with inactivated KV-H5 vaccine at 8 days old), Group III (unvaccinated control).This work was supported by the United States Agency for International Development (USAID) under a grant (AID-263-IO-11-00001, Mod. #3) and within the framework of OSRO/EGY/101/ USA, which applies to projects jointly implemented by the FAO, GOVS and NLQP.http://www.plosone.orgam2016Production Animal StudiesVeterinary Tropical Disease

Distribution pattern of antibiotic resistance genes in Escherichia coli isolated from colibacillosis cases in broiler farms of Egypt

Background and Aim: Multidrug resistance (MDR) of Escherichia coli has become an increasing concern in poultry farming worldwide. However, E. coli can accumulate resistance genes through gene transfer. The most problematic resistance mechanism in E. coli is the acquisition of genes encoding broad-spectrum β-lactamases, known as extended-spectrum β-lactamases, that confer resistance to broad-spectrum cephalosporins. Plasmid-mediated quinolone resistance genes (conferring resistance to quinolones) and mcr-1 genes (conferring resistance to colistin) also contribute to antimicrobial resistance. This study aimed to investigate the prevalence of antimicrobial susceptibility and to detect β-lactamase and colistin resistance genes of E. coli isolated from broiler farms in Egypt. Materials and Methods: Samples from 938 broiler farms were bacteriologically examined for E. coli isolation. The antimicrobial resistance profile was evaluated using disk diffusion, and several resistance genes were investigated through polymerase chain reaction amplification. Results: Escherichia coli was isolated and identified from 675/938 farms (72%) from the pooled internal organs (liver, heart, lung, spleen, and yolk) of broilers. Escherichia coli isolates from the most recent 3 years (2018–2020) were serotyped into 13 serotypes; the most prevalent serotype was O125 (n = 8). The highest phenotypic antibiotic resistance profiles during this period were against ampicillin, penicillin, tetracycline, and nalidixic acid. Escherichia coli was sensitive to clinically relevant antibiotics. Twenty-eight selected isolates from the most recent 3 years (2018–2020) were found to have MDR, where the prevalence of the antibiotic resistance genes ctx, tem, and shv was 46% and that of mcr-1 was 64%. Integrons were found in 93% of the isolates. Conclusion: The study showed a high prevalence of E. coli infection in broiler farms associated with MDR, which has a high public health significance because of its zoonotic relevance. These results strengthen the application of continuous surveillance programs

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Genotyping of rabbit hemorrhagic disease virus detected in diseased rabbits in Egyptian Provinces by VP60 sequencing

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

Detection of aerobic bacterial pathogens associated with early embryonic death in pregnant New Zealand female Rabbits in Egypt

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

Evaluating Flinders Technology Associates card for transporting bacterial isolates and retrieval of bacterial DNA after various storage conditions

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

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019&ndash;2021

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&ndash;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&ndash;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&ndash;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

HI titres^* (mean ±SD) and sero-positivity rates for the selected ducks subject to the four challenge experiments^**.

<p>HI titres^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156747#t002fn002" target="_blank">*</a>} (mean ±SD) and sero-positivity rates for the selected ducks subject to the four challenge experiments^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156747#t002fn003" target="_blank">**</a>}.</p