Cytokine expression, protection and shedding reduction induced by the combination of lipopolysaccharide with Montanoid ISA71 in oil-based Newcastle disease vaccine.

Oil-based inactivated ND vaccines are a commonly used control strategy for this endemic disease in Egypt. One of the major limitations of these inactivated vaccines is the time taken to develop a protective response in vaccinated birds. In the present study, we aimed to formulate an inactivated oil-based ND vaccine incorporated with lipopolysaccharide (LPS) that stimulates the early onset innate response to inactivated vaccines via proinflammatory cytokine production. Five groups of 21-day old SPF chicks were reared in isolators and were treated as follows: G1: Montanoid ISA71 adjuvanted NDV vaccinated group, G2: LPS and Montanoid ISA71 adjuvanted NDV vaccinated group, G3: LPS and Montanoid ISA71 with phosphate buffer saline received group and two non-vaccinated control groups. NDV specific antibodies and cell mediated immune responses were evaluated by hemagglutination inhibition and lymphocyte proliferation tests, respectively. Transcriptional responses of the TLR4, IFN-γ and IL-2 genes were analyzed in peripheral blood mononuclear cells (PBMCs) following vaccination by qRT-PCR. Protection % was determined after challenge with a lethal strain of NDV 106 EID50/0.5 ml. Viral shedding was assessed on oropharyngeal swabs by qRT-PCR and infectivity titration on SPF-ECE. The results revealed that the incorporation of LPS with ISA71 in the oil-based ND vaccine induced a synergistic response confirmed by significant humoral and lymphoproliferative responses with a significant increase in Th1 cytokine transcripts. The simultaneous use of both adjuvants in G2 demonstrated complete protection and a significant reduction in viral shedding compared to the ISA71-adjuvated ND vaccine in G1, which conferred 90 % protection.

Short beak and dwarfism syndrome among Pekin ducks: First detection, full genome sequencing, and immunohistochemical signals of novel goose parvovirus in tongue tissue.

Novel goose parvovirus (NGPV) is continuously threatening the global duck industry, as it causes short beak and dwarfism syndrome among different duck breeds. In this study, we investigated the viral pathogenesis in the tongue of affected ducks, as a new approach for deeper understanding of the syndrome. Seventy-three, 14- to 60-day-old commercial Pekin ducks were clinically examined. Thirty tissue pools of intestine and tongue (15 per tissue) were submitted for molecular identification. Clinical signs in the examined ducks were suggestive of parvovirus infection. All examined ducks had short beaks. Necrotic, swollen, and congested protruding tongues were recorded in adult ducks (37/73, 51%). Tongue protrusion without any marked congestion or swelling was observed in 20-day-old ducklings (13/73, 18%), and no tongue protrusion was observed in 15-day-old ducklings (23/73, 32%). Microscopically, the protruding tongues of adult ducks showed necrosis of the superficial epithelial layer with vacuolar degeneration. Glossitis was present in the nonprotruding tongues of young ducks, which was characterized by multifocal lymphoplasmacytic aggregates and edema in the propria submucosa. Immunohistochemical examination displayed parvovirus immunolabeling, mainly in the tongue propria submucosa. Based on polymerase chain reaction, goose parvovirus was detected in 9 out of 15 tongue sample pools (60%). Next-generation sequencing confirmed the presence of a variant goose parvovirus that is globally named NGPV and closely related to Chinese NGPV isolates. Novel insights are being gained from the study of NGPV pathogenesis in the tongue based on molecular and immunohistochemical identification.

Molecular and pathological characterisation of genotype VII Newcastle disease virus on Egyptian chicken farms during 2016-2018.

Newcastle disease virus (NDV) remains a constant threat to the poultry industry even with intensive vaccination programmes. In the present study, 40 samples were collected from farms showing high mortalities in some Egyptian governorates between 2016 and 2018. Tracheal samples were collected for virus isolation and confirmed by real-time RT-PCR. Molecular characterisation was performed by sequencing, followed by phylogenetic analysis of the novel sequences. Histopathological and immunohistochemical examinations were performed on different organs from NDV-infected broilers. The phylogenetic analysis revealed that the NDV isolates from different areas of Egypt were genetically closely related and all belonged to genotype VII. The histopathological hallmarks included haemorrhagic tracheitis, interstitial pneumonia with syncytia formation, haemorrhagic proventriculitis, necrotising pancreatitis, pan-lymphoid depletion, non-suppurative encephalitis and nephritis. Immunological detection of NDV antigen clarified the widespread presence of viral antigen in different organs with severe lesions. The present study confirmed that a virulent NDV of genotype VII became the predominant strain, causing severe outbreaks in poultry farms in Egypt. The presence of viral antigen in different organs indicates the pantropic nature of the virus. Immunohistochemistry was a very useful diagnostic tool for the detection of NDV antigen.

Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt.

Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.

Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus.

BACKGROUND: Nano-PCR is a recent tool that is used in viral diseases diagnosis. The technique depends on the fundamental effects of gold nanoparticles (AuNPs) and is considered a very effective and sensitive tool in the diagnosis of different diseases including viral diseases. Although several techniques are currently available to diagnose foot and mouth disease virus (FMDV), a highly sensitive, highly specific technique is needed for specific diagnosis of the disease. In the present work, a novel AuNPs biosensor has been designed using thiol-linked oligonucleotides that recognize the conserved 3D gene of FMDV. RESULTS: The AuNPs-FMDV biosensor specifically recognizes RNA standards of FMDV, but not that of swine vesicular disease virus (SVDV) isolates. The analytical sensitivity of the AuNPs-FMDV biosensor was 10 copy number RNA standards in RT-PCR and 1 copy number RNA standard in real-time rRT-PCR with a 94.5% efficiency, 0.989 R2, a - 3.544 slope and 100% specificity (no cross-reactivity with SVDV). These findings were confirmed by the specific and sensitive recognition of 31 Egyptian FMDV clinical isolates that represents the three FMDV serotypes (O, A, and SAT2). CONCLUSIONS: The AuNPs-FMDV biosensor presents in this study demonstrates a superior analytical and clinical performance for FMDV diagnosis. In addition, this biosensor has a simple workflow and accelerates epidemiological surveillance, hence, it is qualified as an efficient FMDV diagnosis tool for quarantine stations and farms particularly in FMDV endemic areas.

SARS-CoV-2 infection of companion animals in Egypt and its risk of spillover.

BACKGROUND: Reverse zoonoses occur because of interactions between humans and animals. Homology of ACE-2 cell receptors in different hosts and high mutation rate of SARS-CoV-2 enhance viral transmission among species. OBJECTIVES: This study aimed to investigate spillover of SARS-CoV-2 between humans and companion animals. METHODS: A cross-sectional study was constructed using nasopharyngeal/oropharyngeal swabs, serum and blood samples collected from 66 companion animals (33 cats and 33 dogs) that were in contact with SARS-CoV-2-positive owners from December 2020 to March 2021. Swabs were screened by rRT-PCR and some positive cases were confirmed by partial spike gene sequencing. Clinical pathology and pathological studies were also performed. RESULTS: Our findings revealed that 30% of cats (10/33) and 24% of dogs (8/33) were SARS-CoV-2 positive. While 33% of these animals were asymptomatic (6/18), 28% showed mild respiratory signs (5/18) and 39% displayed severe respiratory signs (7/18) including 4 dead cats 40% (4/10). Partial spike gene sequencing of 6 positive samples collected in December 2020 were identical to SARS-CoV-2 that was detected in humans in Egypt in that time frame. Clinical pathology findings revealed thrombocytopenia, lymphocytopenia, as well as elevated levels of D-dimer, LDH, CRP, and ferritin. Post-mortem and histopathological examinations illustrated multisystemic effects. CONCLUSIONS: There is a potential occurrence of SARS-CoV-2 spillover between humans and pet animals. IMPACTS: The present study highlighted the potential occurrence of SARS-CoV-2 spillover between humans and their companion animals. Biosecurity measures should be applied to decrease spread of SARS-CoV-2 among humans and pet animals.

Development of multiplex gold nanoparticles biosensors for ultrasensitive detection and genotyping of equine herpes viruses.

Gold nanoparticles (GNPs) biosensors can detect low viral loads and differentiate between viruses types, enabling early diagnosis and effective disease management. In the present study, we developed GNPs biosensors with two different capping agent, citrate-GNPs biosensors and polyvinylpyrrolidone (PVP)-GNPs biosensors for detection of EHV-1 and EHV-4 in multiplex real time PCR (rPCR). Citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-1 with mean Cycle threshold (Ct) 11.7 and 9.6, respectively and one copy as limit of detection, while citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-4 with mean Ct 10.5 and 9.2, respectively and one copy as limit of detection. These findings were confirmed by testing 87 different clinical samples, 4 more samples were positive with multiplex GNPs biosensors rPCR than multiplex rPCR. Multiplex citrate-GNPs and PVP-GNPs biosensors for EHV-1 and EHV-4 are a significant breakthrough in the diagnosis of these virus types. These biosensors offer high sensitivity and specificity, allowing for the accurate detection of the target viruses at very low concentrations and improve the early detection of EHV-1 and EHV-4, leading to faster control of infected animals to prevent the spread of these viruses.

Interspecies transmission of SARS CoV-2 with special emphasis on viral mutations and ACE-2 receptor homology roles.

COVID-19 outbreak was first reported in 2019, Wuhan, China. The spillover of the disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to a wide range of pet, zoo, wild, and farm animals has emphasized potential zoonotic and reverse zoonotic viral transmission. Furthermore, it has evoked inquiries about susceptibility of different animal species to SARS-CoV-2 infection and role of these animals as viral reservoirs. Therefore, studying susceptible and non-susceptible hosts for SARS-CoV-2 infection could give a better understanding for the virus and will help in preventing further outbreaks. Here, we review structural aspects of SARS-CoV-2 spike protein, the effect of the different mutations observed in the spike protein, and the impact of ACE2 receptor variations in different animal hosts on inter-species transmission. Moreover, the SARS-CoV-2 spillover chain was reviewed. Combination of SARS-CoV-2 high mutation rate and homology of cellular ACE2 receptors enable the virus to transcend species barriers and facilitate its transmission between humans and animals.

Mutations of the SARS-CoV-2 Spike Glycoprotein Detected in Cats and Their Effect on Its Structure and Function.

The high frequency of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) mutations and homology of the Angiotensin-Converting Enzyme-2 (ACE2) cell receptors in various hosts help the virus transcend species barriers. In this study, we investigated the mutations of the SARS-CoV-2 spike glycoprotein detected in cats and their effect on its structure and function. Interestingly, some of these mutations are reported here in cats for the first time. Structural analysis showed seven residue substitutions in the spike glycoprotein. Four of the detected mutations are located on the spike surface, which are critical interaction points for neutralizing antibodies. Furthermore, three of the reported mutations could facilitate viral binding to the ACE2 host receptor, influence S1/S2 cleavage, destabilize the ß-hairpin structure of the S2 and enhance viral infectivity. Structural modeling and phylogenic analysis of the ACE2 receptor provided an indication of the binding capacity of the virus to the specific cell receptors of different species and hosts. The presented work highlights the effects of the residue substitutions on viral evasion, infectivity and possibility of SARS-CoV-2 spillover between humans and cats. In addition, the work paves the way for in-depth molecular investigation into the relationship between SARS-CoV-2 receptor binding and host susceptibility.

Pulmonary deregulation of expression of miR-155 and two of its putative target genes; PROS1 and TP53INP1 associated with gold nanoparticles (AuNPs) administration in rat.

Background: Gold nanoparticles (AuNPs) have been considered as an ideal candidate in various biomedical applications due to their ease of tailoring into different size, shape, and decorations with different functionalities. The current study was conducted to investigate the epigenetic alteration in the lung in response to AuNPs administration regarding microRNA-155 (miR-155) gene which can be involved in AuNP-induced lung pathogenesis. Methods: Thirty-two Wister rats were divided into two equal groups, control group and AuNPs treated group which received a single intravenous (IV) injection of plain spherical AuNPs (0.015 mg/kg body wt) with an average diameter size of 25±3 nm. Lung samples were collected from both the control and injected groups at one day, one week, one month and two months post-injection. The alteration of relative expression of miR-155 gene and two of its putative target genes; tumor protein 53 inducible nuclear protein 1 (TP53INP1) and protein S (PROS1) was investigated by real time PCR and protein S (PS) expression was analyzed by Western blotting technique. Results: The obtained results revealed that AuNPs administration significantly increases the expression level of miR-155 and reduce relative mRNA expression of TP53INP1 and PROS1 genes at one day post-injection. In contrast, a significant down-regulation of miR-155 level of expression concurrent with up-regulation of expression level of TP53INP1 and PROS1 genes were shown at one week, one month and two months post-injection. PS levels were mirrored to their PROS1 mRNA levels except for two month post-injection time point. Conclusions: These findings indicate epigenetic modulation in the lung in response to AuNPs administration regarding the miR-155 gene which can be involved in AuNP-induced lung pathogenesis.

Genotypes II and VIId-based inactivated Newcastle disease vaccine reduces virus shedding.

In Egypt, recent outbreaks were reported in NDV-vaccinated flocks. The isolated strain was characterized as class II velogenic genotype VIId of Newcastle disease virus (NDV). In this study, three inactivated NDV vaccine formulations were prepared, the first one is LaSota (genotype II), the second one is genotype VIId and the third one is combined Lasota and genotype VIId at a proportion of 1:1. The challenge trials were conducted in SPF chicks to evaluate the efficacy of the prepared vaccines using 106 EID50/0.5 ml of the Egyptian genotype VIId strain of Newcastle disease virus (NDV-B7-RLQP-CH-EG-12). Our results revealed that all three prepared vaccine formulations conferred 100% protection in the vaccinated chicks. However, the combined vaccine induced the highest haemagglutination inhibition (HI) titers and neutralization indices with significant reduction in virus shedding compared to other vaccine formulations. Histopathology examination of different organs collected from vaccinated chicks post challenge indicated the protective efficacy in vaccinated groups compared to the positive control group where a score of severe lesions was shown. This study reports the efficacy of combined inactivated Lasota and genotype VIId vaccine in reducing virus shedding which is very important in controlling NDV infection in chicken.

First report of fowl adenovirus 8a from commercial broiler chickens in Egypt: molecular characterization and pathogenicity.

This study was performed to isolate fowl adenovirus (FAdV) circulating in commercial meat-type chicken in Egypt during 2015 and to identify the pathogenicity of the isolated virus. Cloacal swabs were collected from 9 commercial broiler farms from chickens of 3-5 wk of age in Behira province in Egypt during 2015. FAdV was isolated on chicken embryo liver cells. The virus was identified by conventional polymerase chain reaction targeting a conserved region in the hexon gene. Moreover, phylogenetic analysis of the L1 loop of the hexon gene revealed that the isolated viruses clustered with reference strains belonging to FAdV serotype 8a. This is the first record of FAdV from Egypt on the GenBank. The isolated virus is closely related to strains directly associated with inclusion body hepatitis (IBH) causing considerable economic losses. Pathogenicity study of the virus did not show any mortality, although necropsy and histopathological examination displayed severe hepatitis and degenerative changes in the immune system after 5 d from infection, proving that the virus can cause IBH with intermittent shedding.

Five BoLA-DRB3 genotypes detected in Egyptian buffalo infected with Foot and Mouth disease virus serotype O.

Foot and Mouth disease (FMD) is a contagious disease leads to economically loss in livestock production all over the world. This serious disease is caused due to the infection of the animal with a single-stranded RNA virus (FMDV). This study aimed to investigate the genetic polymorphism of BoLA-DRB3 gene in Egyptian buffalo as a candidate genetic marker included in multi-factorial process of FMD resistance/susceptibility. Also this work aimed to genetically characterization and serotyping of circulating FMD virus in Egypt during 2016. For serotyping of FMDV, RT-PCR was used for FMDV-positive samples and the results declared the presence of serotype O in all tested animals. The sequence analysis of FMDV samples revealed five different patterns for the detected serotype O which were submitted to GenBank under the accession Nos.: MG017361-MG017365. The 302-bp amplified fragments from BoLA-DRB3 exon 2 were digested with HaeIII endonuclease and the results showed that the presence of five BoLA-DRB3 genotypes, among them the genotype AA might be associated with FMD-resistance (P < 0.01). On the other hand, genotype AC could be correlated with susceptibility (P < 0.01) to FMD in Egyptian buffaloes where it was absent in resistant group. The five detected genotypes of BoLA-DRB3 exon 2 were submitted to GenBank with the accession Nos.: MF977316-MF977320. In conclusion, our findings suggested that the detection of different BoLA-DRB3 genotypes may be has a promising role for raising the resistance of Egyptian buffalo against FMDV especially serotype O which is prevalent in Egypt with preferring genotype AA.

Prime-boost vaccination strategy against avian influenza and Newcastle disease viruses reduces shedding of the challenge viruses.

In the present study, we carried-out assessment of efficacy of different immunization strategies using two bivalent vaccine formulations containing antigens of inactivated Newcastle disease virus (NDV-genotype VIId) and reassortant highly pathogenic avian influenza virus (H5N1-HPAIV) mixed with Montanide ISA71 and Montanide Gel02 as adjuvants. The efficacy of the prepared vaccines was evaluated by determining the cellular and humoral immune responses. In addition, protection against H5N1-AIV and NDV-genotype VIId challenge viruses post vaccination was assessed when Montanide-Gel02 based vaccine was inoculated in 10-days-old specific pathogen free chicks intraocularly once, twice or once followed by a boost with the Montanide ISA71 based vaccine. The cytokines profile analysis demonstrated that the prime-boost strategy induced the highest up-regulation in interferon-gamma (11.39-fold change) and interleukin-6 (14.12-fold change) genes expression. Also, enhanced lymphocytes proliferation was recorded beside increased antibody titers with protection levels reaching 50 and 60% against H5N1 and NDV challenge; respectively. Immunization with Montanide ISA71 inactivated vaccine induced 80% protection; however, the prime-boost combination afforded complete protection (100%) in the challenged chickens against mortality, clinical signs and virus shedding. Finally, these results highlight the significance of considering not only different vaccine platforms but also vaccination strategies to maximize protection against AIV and NDV with regards to the longevity of the vaccine-induced immune response.

Impact of H5N8 duck and chicken viral isolates on the immunological profile of vaccinated specific pathogen free chickens

Nowadays, there is a global concern about outbreaks caused by the highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 which caused devastating losses in the poultry industry sector. This clade was subdivided into two waves: clade 2.3.4.4A from 2014 to 2015 and clade 2.3.4.4b from 2016 until now. In this literature we aimed to evaluate the efficacy of recently used inactivated commercial avian influenza vaccines against two new Egyptian highly pathogenic avian influenza virus H5N8 isolates of clade 2.3.4.4b, A/chicken/Egypt/1526v/2020/H5N8 (H5N8-CH) and A/Duck/Egypt/Qalubia321/2021 (H5N8-D). Three-week-old specific pathogen free chickens were vaccinated with eight types of the most recently used inactivated avian influenza vaccines containing homologous and heterologous virus to the circulating H5N8 isolates. All specific pathogen free chicken groups were bled weekly post vaccination for antibody analysis using two H5N8 isolates of chicken and duck origin as antigen in hemagglutination inhibition test. Also, all vaccinated chicken groups were challenged 4 weeks post vaccination against the H5N8 duck isolate with a dose of 109 EID50/0.1 mL per chicken to measure the protection percentage of the commercial vaccines used. The results showed that vaccines with homologous and heterologous virus showed variable degrees of accepted protection percentage ranged from 90percent to 100percent, thus it was concluded that not only the genetic and antigenic match of the vaccine strains with the circulating highly pathogenic avian influenza viruses influences vaccine efficiency; other factors, such as manufacturing procedures, adjuvant, antigen content, vaccine dose and administration factors could affect vaccine efficacy, therefore, further vaccine development studies are needed to improve the percentage of protection and prevention of viral shedding against local highly pathogenic avian influenza H5 viruses in Egypt(AU)

En la actualidad, existe una preocupación mundial por los brotes causados por el virus de la gripe aviar altamente patógena H5N8 clado 2.3.4.4 que causó pérdidas devastadoras en el sector de la industria avícola. Este clado se subdividió en dos oleadas: clado 2.3.4.4A de 2014 a 2015 y clado 2.3.4.4b de 2016 hasta ahora. En el presente trabajo, dos aislamientos egipcios de la gripe aviar altamente patógena H5N8 del clado 2.3.4.4b, A/chicken/Egypt/1526v/2020/H5N8 (H5N8_CH) y A/Duck/Egypt/Qalubia321/2021 (H5N8_D), se utilizaron para evaluar la eficacia de vacunas comerciales inactivadas contra la gripe aviar de reciente utilización. Pollos libres de patógenos específicos de tres semanas de edad fueron vacunados con ocho vacunas inactivadas contra la influenza aviar, de uso reciente, que contenían virus homólogos y heterólogos a los aislamientos circulantes de H5N8. Todos los grupos de pollos libres de patógenos específicos fueron sangrados semanalmente tras la vacunación para el análisis de anticuerpos; dos virus H5N8 aislados de pollo y pato se utilizaron como antígeno en la prueba de inhibición de la hemaglutinación. Además, todos los grupos de pollos vacunados fueron retados 4 semanas después de la vacunación con el virus H5N8 aislado de pato, con una dosis de 109 EID50/0,1 mL por pollo, para medir el porcentaje de protección de las vacunas comerciales utilizadas. Los resultados mostraron que las vacunas con virus homólogos y heterólogos presentaron grados variables de aceptada protección, la que osciló entre el 90 por ciento y el 100 por ciento, por lo que se concluyó que no sólo la coincidencia genética y antigénica de las cepas vacunales con los virus circulantes de la influenza aviar altamente patógena influye en la eficacia de la vacuna; otros factores, como los procedimientos de fabricación, el adyuvante, el contenido en antígenos, la dosis de la vacuna y los factores de administración podrían afectar a la eficacia de la vacuna, por lo que es necesario seguir estudiando el desarrollo de vacunas para mejorar la protección y la prevención de la excreción viral contra los virus H5 de la influenza aviar altamente patógena locales en Egipto(AU)

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses.

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.

Enhanced protection against FMDV in cattle after prime- boost vaccination based on mucosal and inactivated FMD vaccine.

Improved immunization and control strategies and platforms are greatly needed for foot and mouth disease virus (FMDV) and mucosal vaccines propose an effective strategy for the control FMDV by blocking viral entry. In this study, several immunization strategies, using two FMDV vaccine formulations, including Montanide ISA 206 oil-based FMD inactivated vaccine and Montanide IMS 1313 VG N PR-based concentrated semi-purified FMD mucosal vaccine, were applied. Results of intranasal immunization with the prepared FMD mucosal vaccine, given once or twice, induced IgA levels in both nasal and salivary secretions besides a high response of lymphocyte proliferation with protection levels reaching 20% and 40%, respectively, in a challenge trial in cattle. Immunization with Montanide 206 inactivated FMD vaccine was capable of inducing 80% protection whereas prime-boost strategy based on the administration of mucosal vaccine followed by inactivated vaccine appeared to be the most potent strategy by achieving 100% protection against an FMDV challenge. Indeed, the study reports the efficacy of the prepared IMS 1313 FMD mucosal vaccine and the possible use of this vaccine in the context of different vaccination strategies to control FMDV.

Complete genome sequences of two avian infectious bronchitis viruses isolated in Egypt: Evidence for genetic drift and genetic recombination in the circulating viruses.

Avian infectious bronchitis virus (IBV) is highly prevalent in chicken populations and is responsible for severe economic losses to poultry industry worldwide. In this study, we report the complete genome sequences of two IBV field strains, CU/1/2014 and CU/4/2014, isolated from vaccinated chickens in Egypt in 2014. The genome lengths of the strains CU/1/2014 and CU/4/2014 were 27,615 and 27,637 nucleotides, respectively. Both strains have a common genome organization in the order of 5'-UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-UTR-poly(A) tail-3'. Interestingly, strain CU/1/2014 showed a novel 15-nt deletion in the 4b-4c gene junction region. Phylogenetic analysis of the full S1 genes showed that the strains CU/1/2014 and CU/4/2014 belonged to IBV genotypes GI-1 lineage and GI-23 lineage, respectively. The genome of strain CU/1/2014 is closely related to vaccine strain H120 but showed genome-wide point mutations that lead to 27, 14, 11, 1, 1, 2, 2, and 2 amino acid differences between the two strains in 1a, 1b, S, 3a, M, 4b, 4c, and N proteins, respectively, suggesting that strain CU/1/2014 is probably a revertant of the vaccine strain H120 and evolved by accumulation of point mutations. Recombination analysis of strain CU/4/2014 showed evidence for recombination from at least three different IBV strains, namely, the Italian strain 90254/2005 (QX-like strain), 4/91, and H120. These results indicate the continuing evolution of IBV field strains by genetic drift and by genetic recombination leading to outbreaks in the vaccinated chicken populations in Egypt.

Comparative Effectiveness of Two Oil Adjuvant-Inactivated Avian Influenza H9N2 Vaccines.

Low pathogenic avian influenza H9N2 virus infection has been an important risk to the Egyptian poultry industry since 2011. Economic losses have occurred from early infection and co-infection with other pathogens. Therefore, H9N2 vaccination of broiler chicks as young as 7 days old was recommended. The current inactivated H9N2 vaccines (0.5 ml/bird) administered at a reduced dose (0.25 ml/bird) do not guarantee the delivery of an effective dose for broilers. In this study, the efficacy of the reduced-dose volume (0.3 ml/bird), compared with the regular vaccine dose (0.5 ml/bird) of inactivated H9N2 vaccines using two different commercially available adjuvants, was investigated. The vaccines were prepared from the local H9N2 virus (Ck/EG/114940v/NLQP/11) using the same antigen content: 300 hemagglutinating units. Postvaccination (PV) immune response was monitored using the hemagglutination inhibition test. At 4 wk PV, both vaccinated groups were challenged using the homologous H9N2 strain at a 50% egg infective dose (EID50) of 10(6) EID50/bird via the intranasal route. Clinical signs, mortality, and virus shedding in oropharyngeal swabs were monitored at 2, 4, 6, and 10 days postchallenge (DPC). The reduced-dose volume of vaccine induced a significantly faster and higher immune response than the regular volume of vaccine at 2 and 3 wk PV. No significant difference in virus shedding between the two vaccine formulas was found (P ≥ 0.05), and both vaccines were able to stop virus shedding by 6 DPC. The reduced-dose volume of vaccine using a suitable oil adjuvant and proper antigen content can be used effectively for early immunization of broiler chicks.

A Dose-Response Study of Inactivated Low Pathogenic Avian Influenza H9N2 Virus in Specific-Pathogen-Free and Commercial Broiler Chickens.

Since the first report of low pathogenic avian influenza (LPAI) H9N2 virus in Egypt in 2011, the Egyptian poultry industry has suffered from unexpected economic losses as a result of the wide spread of LPAI H9N2. Hence, inactivated H9N2 vaccines have been included in the vaccination programs of different poultry production sectors. The optimal antigen content of avian influenza virus vaccines is essential to reach protective antibody titers. In this study, the correlation between antigen content (based on hemagglutinating units [HAU]) and postvaccination (PV) antibody response of H9N2 inactivated vaccine was studied. Five different vaccine antigen loads (128, 200, 250, 300, and 350 HAU formulas/dose) were investigated in commercial broiler and specific-pathogen-free (SPF) chickens. Vaccine safety and PV antibody responses were monitored. At the fourth week PV only SPF vaccinated groups (128, 200, 250, and 300 HAU/dose) were challenged using LPAI H9N2 (A/Ck/EG/114940v/NLQP/11) virus with 10(6) EID50/bird. Oropharyngeal swabs were used to monitor virus shedding at 2, 4, 6, and 10 days postchallenge. Results showed that all vaccine formulations were well tolerated, and the highest antibody titers were observed in birds vaccinated with higher HAU. Vaccines containing 128 and 200 HAU/dose did not induce the required protective HI titers by 3 wk PV. Meanwhile, the challenge experiment in SPF chickens showed that 250 and 300 HAU vaccine doses were required to reduce the level and duration of virus shedding. Study results thus suggest that inactivated H9N2 vaccines containing at least 250 HAU/dose will induce the optimal protective titers and minimize virus shedding in SPF chickens.