Experimental trials to assess the immune modulatory influence of thyme and ginseng oil on NDV-vaccinated broiler chickens.

Background: The use of traditional medicine against viral diseases in animal production has been practiced worldwide. Herbal extracts possess organic substances that would improve chicken body performance. Aim: The current study was designed to evaluate the effect of either thyme or ginseng oil in regard to their immune-modulatory, antiviral, and growth promoter properties. Methods: Two hundred and forty-one-day-old broiler chicks were allocated into eight equal groups as the following: group 1; nonvaccinated and nontreated and group 2; Newcastle disease virus (NDV) vaccinated and nontreated. Birds of groups 3 and 4 were treated with thyme oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 5 and 6 were treated with ginseng oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 7 and 8 were treated with a combination of ginseng oil (100 mg/l of drinking water) and thyme oil (100 mg/l of drinking water) for 12 hours/day. On the 35th day of life, birds in all the experimental groups were given 0.1 ml of a virulent genotype VIId NDV strain suspension containing 106.3 EID50/ml intramuscularly. Results: Administration of ginseng and thyme oils each alone or simultaneously to birds either vaccinated or nonvaccinated elicited a significant improvement in body performance parameters. Administration of thyme and ginseng each alone or concurrently to vaccinated birds (Gp 4, 6, and 8) induced a higher hemagglutination inhibition (HI) titer of 6, 7.3, and 6.3 log2 at 21 days of age, 6.7, 7.6, and 7 log2, at 28 days of age and 7, 8, and 6.8 log2 at 35 days of age, respectively. Challenge with vNDV genotype VII led to an increase in the NDV-specific HI-Ab titers 10 days post challenge in all the experimental groups. In addition, thyme, ginseng oils, or a combination of them improved the protection from mortality in vaccinated birds; by 100%, 100%, and 90%, respectively, compared with 80% protection from mortality in vaccinated-only birds post-NDV challenge. Moreover, NDV-vaccinated birds treated either with thyme; ginseng or their combination showed negative detection of the virus in both tracheal and cloacal swabs and nonvaccinated groups that received oils showed improvement in vNDV shedding in tracheal and cloacal swabs. Conclusion: It could be concluded that the administration of thyme and ginseng essential oils to broilers can improve productive performance parameters, stimulate humoral immunity against, and protect from vNDV infection.

Evaluation of the immuno-stimulatory effect of aqueous neem (Azadirachta indica) leaf extract against highly pathogenic avian influenza (H5N8) in experimental chickens.

The recently detected clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) H5N8 virus in poultry encouraged us to study the efficacy of the 6 most extensively used saleable H5 poultry vaccinations (bivalent [AI + ND], Re-5 H5N1, H5N1, H5N3, monovalent AI, monovalent ND) with or without aqueous 8% neem (Azadirachta indica) leaf extract as an immunostimulant. One hundred thirty birds were randomly divided into 7 groups. Groups 1, 2, 3, 4, 5, and 6 were divided into 2 subgroups (G1a, G2a, G3a, G4a, G5a, G6a) and (G1b, G2b, G3b, G4b, G5b, G6b) with 10 birds each. Subgroups (G1a, G2a, G3a, G4a, G5a, G6a) received the (bivalent [AI + ND], Re-H5N1, H5N1, H5N3, monovalent AI, monovalent ND) vaccines, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination but treated with neem leaf extract administrated 2 d before and after vaccination, and G7 with 10 birds was kept unvaccinated as positive control group. Clinical signs of the challenged group showed conjunctivitis, closed eyes, cyanosis in comb and wattle, ocular discharge, and greenish diarrhea, while postmortem lesions showed congested trachea and lung, hemorrhage on the shank, proventriculus, and pancreas; gelatinous fluid submandibular, congestion of all organs (septicemia), mottled spleen. The clinical signs and lesions were mild in neem leaf extract treated with bivalent vaccine and Re-H5N1 while moderate in monovalent vaccine and H5N3 with or without neem leaf extract treated and reached severe in the group immunized with H5N1 with or without neem leaf extract treatment. The protection levels in the bivalent vaccine (AI + ND), Re-5 H5N1, and H5N3 treated with neem leaf extract, were 80%, 80%, and 60%, respectively, while bivalent vaccine (AI + ND), Re-5 H5N1 and H5N3 without treatment were 60%, 60%, and 40%, respectively. The virus shedding was prevented in groups vaccinated with bivalent vaccine and Re-H5N1 vaccine treated with neem leaf extract, while decreased in the group vaccinated with H5N3 with neem leaf extract and Re-H5N1 without neem leaf extract compared with H5N3, H5N1, and monovalent vaccine. The immunological response after vaccination was stronger in the bivalent vaccine group than in the other commercial vaccine groups treated with neem leaf extract, with geometric mean titer (GMTs) of 315.2 and 207.9 at the third and fourth weeks, respectively. The use of immunostimulant antiviral medicinal plants, such as neem, completely protected chicken flocks against HPAI (H5N8) and prevented AI virus shedding, leading us to the conclusion that the use of bivalent vaccines induces a higher immune response than other different commercial vaccines.

Mycoplasmosis in Poultry: An Evaluation of Diagnostic Schemes and Molecular Analysis of Egyptian Mycoplasma gallisepticum Strains.

Infections with Mycoplasma gallisepticum (MG) in poultry are associated with a wide range of disease conditions, including those affecting the respiratory and reproductive systems. The purpose of this study was to endorse the more sensitive diagnostic scheme for MG infection and identify the best molecular marker for MG phylogenetic analysis using six housekeeping genes: mgc2, mraW, atpG, ugpA, DUF31196, and lgT. For these purposes, 55 poultry flocks of different species were screened using either qRT-PCR or PCR techniques analogous to conventional culturing from non-cultured and cultured swabs on PPLO broth. The rate of MG positivity was the highest when using qRT-PCR from cultured broth (89.0%) and the lowest when using conventional culturing (34.5%). Compared to qRT-PCR from broth, statistical analysis using the Roc curve in MedCalc statistical software showed that the PCR schemes (qRT-PCR from swabs and PCR from swabs and broth) performed better than conventional culturing in terms of sensitivity, accuracy, and area under the curve (AUC), suggesting that they may be more reliable schemes. Further support was added by Cohen's kappa test, showing moderate agreement between the molecular approaches. Among the six screened genes, mgc2 and mraW had the highest detection rates (69% and 65.4%, respectively). The comparative phylogenetic analysis revealed that mgc2 or atpG gene sequences distinguished MG isolates into different clades with high discriminatory power.

The protection conferred against virulent Newcastle disease virus (vNDV) genotype VII by commercial double recombinant HVT vaccines and NDV live-attenuated vaccine as prime/boost vaccination regimens in commercial broiler chickens carrying maternally-derived antibodies (MDAs) against NDV.

Vaccines against vNDV are readily available and potentially protective; nevertheless, improved vaccination protocols are required to prevent clinical disease and discontinue the spread of the virus. This study assessed the effectiveness of two commercial recombinant herpesvirus of turkey vector vaccines (rHVT-NDV-IBDV) that express the fusion (F) protein of NDV and the virus protein 2 (VP2) of infectious bursal disease virus (IBDV). In commercial broilers with maternally-derived antibodies (MDAs) the efficacy of the rHVT-NDV-IBDV vaccines was evaluated when administered alone, in combination with live-attenuated NDV vaccine at one-day-old, or as part of a prime/boost strategy. The vaccinated birds were challenged with the genotype VIId vNDV strain (NDV/chicken/Egypt/1/2015) at various ages (14, 24 and 35 days). In comparison to sham-vaccinated control birds, the applied vaccination regimens were able to reduce or prevent mortality and virus shedding and clinical disease. Two weeks post-application, the two vector vaccines were serologically reactive with the MDAs and induced protective immune responses against the F protein. In the instance of early challenge at 14 days old, the combination of recombinant rHVT-NDV-IBDV with a live vaccine offered better protection and reduced virus shedding compared to the vector vaccine alone. Boosting with live NDV vaccine at 14 days old increased the protective effect of the vector vaccines and reduced virus shedding and the clinical index after challenge at 24 days old. Both combining and/or boosting with live vaccine together with the vector vaccine provided better protection and minimized virus shedding compared with vaccination with vector vaccine only in the instance of 5-week-old challenge.

Newcastle Disease Genotype VII Prevalence in Poultry and Wild Birds in Egypt.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019-2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70-99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

Different kinetics of chicken interferon-alpha signalling transduction responses following immunization of broiler chickens with different Newcastle disease virus vaccines and infection with virulent genotype VIId strain.

Newcastle disease virus (NDV) is a highly contagious and notifiable avian disease leading to grave economic losses in the poultry industry. Although the immune responses against NDV have been widely investigated, little is known regarding the virus interaction with the host innate immune responses. In this study, we tested the effect of different commercially applied Newcastle disease vaccines as well as virulent NDV genotype VIId on the expression pattern of the upstream regulator and downstream effector genes related to chicken interferon-alpha (chIFNα) signalling transduction pathway. Using quantitative real-time PCR analysis, mild transient induction of chIFNα-inducible genes was detected in bird spleen 72 h post-vaccination (hpv) with either live LaSota (respiratory) or VG/GA (enteric) strains. Vaccination with the enteric VG/GA strain led to stimulation of the investigated pathway as early as 24 hpv which continued up to 7 days in bird caecal tonsils. Subcutaneous injection with inactivated LaSota oil adjuvant-based vaccine led to continual stimulation of the investigated pathway up to 7 days post-vaccination (dpv). The recombinant herpesvirus of turkey (rHVT) - NDV vaccine led to remarkable stimulation of all the tested cytokines up to 17 dpv in comparison with LaSota and VG/GA NDV vaccines. Stronger but transient activation of all the tested cytokines was detected in spleens during the first 24 h post-challenge with virulent NDV (vNDV) which reduced gradually and diminished later due to the virus-induced lymphocytic depletion. This study will aid in the discovery of new approaches to control NDV.

In vitro and in vivo experimental trials to assess the modulatory influence of ß-caryophyllene on NDV replication and immunopathogenesis.

A trial was conducted to evaluate the antiviral activity and immunomodulatory effect of B-Caryophyllene (BCP) using NDV as a viral model. First, an in ovo experiment was conducted to estimate the antiviral mechanism of BCP. Next, an in vivo experiment was designed to confirm its antiviral efficacy as well as its immunomodulatory and growth promoting ability. According to the in ovo experiment, BCP possesses antiviral influence up to 61.7% when treated before or during NDV infection. Oral supplementation of chickens with two doses of BCP (200 and 400 µg/bird) resulted in a significant increase in the NDV HI-Ab responses and a significant increase in interferon-α signaling cytokines. These obvious immunomodulatory effects improved the bird clinical protection against virulent NDV challenge. To conclude, we introduced a new compound for the poultry industry sector that has antiviral and immunostimulant properties when supplemented orally before or during NDV infection.

Comparative study on dynamic and immunopathology of four intermediate-plus infectious bursal disease (IBD) vaccines in commercial broiler chickens.

INTRODUCTION: The selection of the right IBD control strategy is primarily based on the choice of the appropriate vaccine strain. High maternal IBD-specific antibodies (Abs) compete with the efficacy IBD vaccine, which necessitates the application of intermediate-plus vaccine strain. METHODS: A comparative experimental study was designed for evaluation of four different commercially available intermediate-plus IBD vaccines in commercial broilers before complete weaning of IBD-specific maternal Abs. RESULTS: As determined by IBD- specific quantitative real-time polymerase chain reaction, three tested vaccine strains (228E, Winterfield H2512, and Winterfield 2512) were able to establish in the bursal tissues as early as six hours (hrs) post-vaccination (PV). Both the 228E and the Winterfield H2512 strains vaccinated groups had the highest viral load and replication rate in the bursal tissues at 24, 36, 48 and 72 hrs PV. Earlier seroconversion, 7-14 days PV, was observed in the case of Winterfield H2512, 228E, and Winterfield 2512 vaccinated birds compared to the Lukert vaccinated birds. The 228E strain was more virulent and induces the highest lesion score with severe degrees of lymphocyte depletion and necrosis which persisted up to 28 days PV. CONCLUSION: Overall, the different intermediate-plus IBD strains possess variable early kinetics in the bursal tissues and eliciting antibody (Ab) responses differently withdifferent degrees of bursal lesions. The assessment of the intrabursal vaccine load together with humoral immunity and bursal damage lesion score are fundamental parameters in the evaluation of the intermediate-plus IBD vaccines.

Influence of Marek's disease virus vaccines on chicken melanoma differentiation-associated gene 5-dependent-type I interferon signal transduction pathway with a highlight on their secondary impact on the immune responses post Newcastle disease virus vaccination.

Marek's disease virus (MDV) leads to a lytic infection of B-lymphocytes in chickens, and also latently infects T-lymphocytes. Although Marek's disease vaccines have been widely in use, little is known about the innate immune response of this important livestock vaccine. In this study, we tested the effect of different commercially applied Marek's disease vaccines on the expression pattern of selected genes related to chicken interferon-alpha (chIFN-α) (melanoma differentiation associated gene 5 "MDA5″ dependent) signal transduction pathway. Both MDV serotype I (Rispens) and serotype III (Herpesvirus of turkey "HVT") vaccines could stimulate MDA5 dependent-type I interferon response as early as three days post vaccination in a dose-dependent manner. The stimulation continued up to 10 days in the instance of HVT vaccine and declined in the case of Rispens. Surprisingly, increasing the doses of the two vaccines led to dose-dependent down-regulation in the expression pattern of the investigated pathway, five and ten days post vaccination. Additionally, to shed the light on the consequent effect on the immune responses of the other viral vaccine, another experimental model based on Newcastle disease virus (NDV) vaccines was designed using HVT, HVT-VP2 and Rispens MDV vaccines. The three MDV vaccines were found to reduce chicken humoral immune response post NDV vaccination. However, only Rispens and HVT-VP2 had suppressive effects on the expression of MDA5-dependent-chIFN-α related cytokines. Consistent with this finding, the protection rate and NDV- humoral immune response post challenge with virulent NDV strain was lower in case of Rispens and HVT-VP2 vaccines.

Effectiveness of different avian influenza (H5) vaccination regimens in layer chickens on the humoral immune response and interferon-alpha signalling immune marker.

Avian influenza (AI) vaccines are widely used to control and eliminate the ongoing avian influenza virus epidemic in Egypt. A strict vaccination policy with inactivated AI vaccines has been widely applied, however the virus still circulating, evolving and causing great negative impact to the poultry sector in Egypt. Therefore, an updated poultry vaccination policy using different vaccine technologies might be valuable as an innovative additional control strategy of AIV in Egypt. In the present study, the effectiveness of different avian influenza (AI) vaccination schedules was evaluated in 300 commercial layer chicks (ISA White) using either the oil-emulsion baculovirus-H5-prototype vaccine (baculovirus-H5 prototype) or turkey herpesvirus (HVT) vector vaccine containing the hemagglutinin (HA) gene from H5N1 strain (rHVT-H5), applied alone or in combination and in different settings. Vaccination with either two injections of the baculovirus-H5 prototype, a single injection of rHVT-H5 or priming with rHVT-H5 at 1 day old followed by boosting with the baculovirus-H5 prototype induced AI-HI protective antibody responses starting as early as 3 to 4 weeks of age and lasting up to the end of the rearing period (16 weeks). A single vaccination with the baculovirus-H5 prototype did not generate a protective antibody titre for the entire rearing period. Furthermore, the present study elucidated that vaccination once or twice with the baculovirus-H5 vaccine prototype activated the chicken interferon-alpha (Ch-IFN-alpha) signalling pathway via transduction of antiviral components, e.g., Mx1 and IRF7. Birds immunized once with rHVT-H5 at 1 day old did not show activation of the Mx1 and IRF7 transcripts; however, following boosting with the baculovirus-H5 prototype vaccine, up-regulation of Mx1 and IRF7 was observed. Based on our findings, it can be concluded that either reinforcement with two injections of the baculovirus-H5 prototype or prime-boost vaccination (rHVT-H5 at 1 day old followed by the baculovirus-H5 prototype vaccine at 8 days old) is a successful strategy to induce both innate and humoral immune responses and could be recommended for the layer production sector over the entire rearing period, especially in AI-endemic areas.

Positive regulation of humoral and innate immune responses induced by inactivated Avian Influenza Virus vaccine in broiler chickens.

Avian Influenza (AI) vaccines are widely used for mammals and birds in a trial to eliminate the Avian Influenza virus (AIV) infection from the world. However and up till now the virus is still existed via modulation of its antigenic structure to evade the pressure of host immune responses. For a complete understanding of the immune responses following AI vaccination in chickens, the modulations of the chickens humoral immune responses and interferon-alpha signaling pathway, as a fundamental part of the innate immune responses, were investigated. In our study, we measured the humoral immune response using hemagglutination-inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests. In addition, chicken interferon-alpha pathway components was measured at RNA levels using Quantitative Real-time PCR (qRT-PCR) following one dose of inactivated H5N1 influenza vaccine at 14 days of age. In this study, the protective levels of humoral antibody responses were observed at 14, 21 and 28 days following immunization with inactivated (Re-1/H5N1) AI vaccine. In the chicken spleen cells, up regulation in the chicken interferon-alpha pathway components (MX1 & IRF7) was existed as early as 48 h post vaccination and remained until 28 days post vaccination at the endogenous state. However, after the recall with ex-vivo stimulation, the up regulation was more pronounced in the transcriptional factor (IRF7) compared to the antiviral gene (MX1) at 28 days post vaccination. So far, from our results it appears that the inactivated H5N1 vaccine can trigger the chicken interferon-alpha signaling pathway as well as it can elicit protective humoral antibody responses.

Effects of florfenicol on the immune responses and the interferon-inducible genes in broiler chickens under the impact of E. coli infection.

Florfenicol (FFC) as a chloramphenicol's derivative is a special broad-spectrum antibiotic that was used in veterinary clinics. In the present study, we investigated the effect of different doses of FFC on the humoral immune response of broiler chickens to Newcastle disease virus (NDV) vaccine under the impact of E. coli infection. In addition, the expression of the interferon-inducible genes (IRF7, 2'-5'OAS and Mx1) were analyzed in the spleen tissue of these chickens using quantitative real-time PCR (qRT-PCR). The non-treated group with FFC and non-infected with E. coli had the highest immune responses against NDV compared with the FFC treated groups. In the case of E. coli infection, the group treated with FFC (30 mg/Kg BWt) showed lower NDV HI and IgG ELISA Ab levels compared to the group treated with FFC (60 mg/Kg BWt). A dose dependent up-regulation was observed in the level of the interferon-alpha pathway related genes (IRF7 and 2'-5'OAS) in the FFC treated groups compared to the non-treated group. At the slaughter time, the numbers of adipocyte in the bone marrow were significantly higher with moderate atrophy of the hematopoietic lineages in the FFC treated birds compared to the non-treated birds. These results indicated that this FFC dosage dependent increase in the humoral immune responses against NDV vaccine could be attributed to its efficient therapeutic effect on the E. coli infection. However, the increase in the FFC dosage can negatively but temporarily affect the chicken body weights. Additionally, it can exert up regulation effect on the chicken innate immune response with moderate hypoplasia of the bone marrow cells.

Molecular characterization and phylogenetic analysis of Marek's disease virus from clinical cases of Marek's disease in Egypt.

In spite of the intensive vaccination policy against the Marek's disease virus (MDV) in Egypt, the Egyptian poultry flocks are still suffering from several tumor and paralysis cases. To investigate if MDV is the possible cause, feather follicle and tumor samples were collected during 2011 from 30 vaccinated chicken flocks experiencing nervous signs, emaciation, and tumor lesions. The samples were screened by PCR amplification of the meq full-length gene. Only five of 30 flocks were positive for MDV. Additionally, we sequenced meq from five samples and gL and gC from three samples. A phylogenetic tree was constructed using the deduced amino acid sequences of the meq gene. The sequence analysis revealed that most of the studied sequences showed > or = 98% identity to the very virulent European ATE and C12/130 isolates and the very virulent Chinese LMS, YA, WS03, and GX070060 MDV isolates. The two glycoproteins, gL and gC, displayed high similarity to the classical MDV strains published in the database regardless of their virulence.

Detection and molecular characterization of avipoxviruses isolated from different avian species in Egypt.

Six clinical cases of avipoxvirus (APV) infection were investigated and molecular biologically studied. The samples were collected from different domesticated birds reared in the Egyptian backyard management system and were propagated on the chorioallantoic membrane of embryonated chicken eggs. The virus isolation was confirmed via PCR amplification of fpv167 (P4b) gene locus. All the studied isolates were characterized as Fowlpox-like viruses based on the amplicon length of fpv140 gene locus. The phylogenetic analysis of fpv167 (P4b) gene clustered Elsharqyia_FWPV1, Elsharqyia_FWPV2, Elsharqyia_FWPV3, Elsharqyia_FWPV4, and Elsharqyia_TKPV strains within subclade A1. Furthermore, Elsharqyia_PGPV strain was clustered within subclade A2 (Turkeypox virus) and showed 100 % nucleic acid identity with the wood pigeon Indian which was isolated in 2009. On the other hand, when the fpv140 gene was used for the phylogenetic analysis, Elsharqyia_PGPV was clustered within subclade A4 (Pigeonpox virus) with the other PGPVs. This study is considered the first molecular record for APVs circulating in the Egyptian birds. Further studies in a larger scale need to be developed to have a better understanding about the molecular characterization of the Egyptian APV strains.