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Phoenix dactylifera, mentha piperita and montanide™ ISA-201 as immunological adjuvants in a chicken model
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Phoenix dactylifera, Mentha piperita and MontanideTM ISA-201 as Immunological Adjuvants in a chicken model Kamelia M. Osman ConceptualizationSupervisionWritingReviewing and Editing , Osama E Kamal Data curationMethodology and Writing original draft preparation , Heba N. Deif Data curationMethodology and Writing original draft preparation , Marwa M Ahmed Writing original draft PII: DOI: Reference:
S0001-706X(19)31447-0 https://doi.org/10.1016/j.actatropica.2019.105281 ACTROP 105281
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
18 October 2019 20 November 2019 20 November 2019
Please cite this article as: Kamelia M. Osman ConceptualizationSupervisionWritingReviewing and Editing , Osama E Kamal Data curationMethodology and Writing original draft preparation , Heba N. Deif Data curationMethodology and Writing original draft preparation , Marwa M Ahmed Writing original draft , Phoenix dactylifera, Mentha piperita and MontanideTM ISA-201 as Immunological Adjuvants in a chicken model, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105281
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Phoenix dactylifera, Mentha piperita and Montanide™ ISA-201 as Immunological Adjuvants in a chicken model
Kamelia M. Osman 1*, Osama E Kamal3, Heba N. Deif 1, Marwa M Ahmed3
1*
Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt
2
Department of Community Health, National University, San Diego, CA USA
3
Veterinary Serum and Vaccine Research Institute, Ministry of Agriculture, Egypt
*Corresponding author, e-mail: [email protected]
1
Abstract This study evaluated plant-based immune-adjuvants from crude extracts of Phoenix dactylifera and Mentha piperita as promising adjuvants for vaccines because of the limited side effects associated with plant extracts. In addition, Montanide™ ISA 201 previously used in vaccines in cattle. Eight different infectious coryza (IC) vaccines were prepared from three serovars [A (W strain and local strain), C (Modesto strain) and B (0222 strain)] with eight Avibacterium. paragallinarum vaccines adjuvants formulae using liquid paraffin, Montanide™ ISA 71, Montanide™ ISA 201, and Montanide™ Gel adjuvants, P. dactylifera and M. piperita as immune-stimulants at a concentration of 1 mg and 2 mg incorporated with or without liquid paraffin oil as an adjuvant. These vaccines were applied in a chicken model. After a single immunization, the eight vaccine formulations were evaluated using the ELISA and Microplate agglutination test. Evidence of protection in the immunized birds was based on the results after challenge and bacterial isolation. The incorporation of the crude aqueous extract of P. dactylifera or M. piperita at a concentration of 2 mg in a liquid paraffin oil adjuvanted IC vaccine could be employed as an efficient adjuvant for chicken to IC vaccine to enhance immune responses. Also,Montanide™ ISA 201 may be the best adjuvant to be used to enhance the protective response against Av. paragallinarum. Our results confirm that aqueous extracts of M. piperita leaves and P. dactylifera fruit have immunomodulatory potentials in vivo and elevated serum antibodies against Av. Paragallinarum. Keywords: P. dactylifera and M. piperita extracts adjuvants; Av. Paragallinarum; Montanide™ ISA-201; ISA-71 and Montanide™ Gel
2
Montanide™ is a family of oil-based adjuvants that have been used in trial vaccines in human and animals (Dar et al. 2013). To date, the adjuvant potential of ISA-201 for Avibacterium paragallinarum poultry vaccine, has not been previously reported. Immunostimulators are substances (e.g. plant extracts) that stimulate the immune system by inducing activation or increasing activity of any of its components (Catanzaro et al. 2018). The most important advantage of plant extracts is that they have minimal adverse side-effects, since they are biodegradable compounds (Kharissovaet al. 2019). Additionally, they are easy to extract, relatively safe, inexpensive, often available (Mastelic et al. 2010). Avian infectious coryza (IC) is an acute upper respiratory tract infection in chickens that is caused by the Gram-negative bacterium Av. Paragallinarum (Ruiz et al., 2018). It is believed to be evolving as a re-emerging worldwide disease of poultry that affects all ages of chickens of both indigenous and laying hens in poultry farms(Gallus gallus domesticus), in pheasants (Phasianus) and guinea fowls (Numida) and Japanese quails (Coturnix japonica) (Brash et al. 2013; Thenmozhi and Malmarugan 2013; CABI,, 2019). Therefore, the present study was undertaken to compare the serological responses and protective efficacy of an inactivated trivalent IC vaccine formulated with a local strain and two international strains of Av. paragallinarum in Montanide™ ISA-201, ISA-71 and Montanide™ Gel in poultry. Also, this study illustrates the enhancing effect of the aqueous extracts P. dactylifera or M. piperita as immunostimulants for IC vaccines. A total of 550 eight-week old SPF white leghorn birds were used. Local field strain according to Page serovar A was originally isolated from an outbreak of IC in a laying flock in Egypt, confirmed at the species level and serotyped using a Page scheme of serotyping with antisera against reference serovars (Blackall et al. 1990). The reference strain 0222 (Page serovar B) was obtained from Dr. R. B. Rimler, USDA National. Animal Disease Center, Ames, Iowa, USA. The reference strains W (page serovar A) and Modesto (Page serovar C) were obtained from MSD Animal Health/Intervet International bv., Boxmeer, The Netherlands. Preparation of crude aqueous extracts of P. dactylifera (date or date palm) and M. piperita (peppermint) leaves were prepared in cold distilled water (1:3 ratio, weight to volume) and kept for 48 hours at a temperature of
with continuous stirring The extracts of both P.
dactylifera and M. piperita were freeze-dried to give a yield of 12.47 g. This was reconstituted separately in distilled water to give the required doses of 1 or 2 mg/kg body weight of the extract used in this study. Av. paragallinarum strains [(Av. paragallinarum references strains (W strain of serovar A, Modesto strain serovar C and 0222 strain of serovar B) as well as a local strain of Av. paragallinarum (serovar A) were inoculated into cBHI medium for 24 h at 37°C. The strains were inactivated with 0.25% formalin, then washed with normal saline. The bacterin of each 3
strain of Av. paragallinarum was adjusted to 6.0× 108cfu/ ml of serovar A (W strain), 1.0 ×109cfu/ml of serovar B (0222 strain) and 8.0 × 108cfu/ ml of serovar C (Modesto strain) as well as 6.0 ×108cfu/ ml of local strain (serovar A). Experimental polyvalent vaccine was adjuvenated with various adjuvants as outlined in Tables S1 and S2. The procedures of vaccine emulsification were carried out according to the SEPPIC vaccine department laboratory, France (www.seppic.com). At 10 weeks of age, the chickens were inoculated using one experimental vaccine formula for each group using the same vaccine code (V-1; V-2; V-3, V-4, V- 5, V- 6, V-7 and V-8). As a preliminary step, all birds were monitored for antibody titres against Av. paragallinarum by the Microplate agglutination test (MAT) (Wambura 2010) and Enzyme-linked immunosorbent assay (ELISA) (Sakamoto et al. 2012). At week 3 post immunization, each bird was challenged once by topical administration into the infra-orbital eye area, consisting of 100 μl (~108 CFU) of the bacterial broth reconstituted in PBS, pH 7.0 (Gong et al. 2013). All chickens were kept under observation for 10 days post challenge. The clinical signs in the birds were recorded during the course of the experiment and scored daily in numerical values relative to the severity of the clinical signs observed (Boucher et al. 2014). The variations within and between groups were analyzed using a One-Way ANOVA followed by Tukey-Kramer multiple comparison test. Minitab 17 Statistical Software (State College, PA: Minitab, Inc.) was used for all statistical analysis. Generally, all chickens in the four groups (V-1, V-2, V-3 and V-4) immunized with the four vaccine formulations developed MAT and ELISA antibodies against each of the four tested strains of Av. Paragallinarum (Tables S1 and S2) which continued to increase at equivalent rates post-immunization, to gradually decline to reach their lowest levels by the 24th week The Montanide™ ISA 201- adjuvant vaccine formulation generated a significantly (P < 0.05) higher rate of Ab titres. The data clearly indicated that using of 2 mg of either P. dactylifera or M. piperita included in the vaccine induced higher levels of antibody titers than when they were used at a concentration of 1 mg. It should also be emphasized that, when the P. dactylifera or M. piperita were used at a concentration of either 1 mg or 2 mg and included in the liquid paraffin oil adjuvant IC vaccine as immunostimulants, they resulted in a better immune response than when the liquid paraffin oil IC vaccine was used alone. Statistically, the incorporation of either 2 mg P. dactylifera or M. piperita with liquid paraffin oil adjuvant IC vaccine, generated a significant (P<0.05) higher rate of antibody titers against the four antigens of Av. paragallinarum than using vaccine with 1 mg of each immunostimulant or the vaccine without the immunostimulants. The protective efficacy of the eight vaccine formulations after challenges are summarized in Table S3. Vaccine emulsification in adjuvants, which potentiate immune responses, are a target area of vaccine development to improve vaccine efficacy (Kaurav 4
et al. 2018). The results suggest the V-3 vaccine formulation which contain Montanide™ ISA 201 could be the best adjuvant to enhance the protective response of the candidate vaccines against Av. paragallinarum. Also, the use of immunostimulators (such as plant extract) with a vaccine can stimulate the immune system by inducing activation or increasing activity of any of its components (Mastelic et al. 2010; Yang et al.. 2018). Medicinal plants which are used as immunomodulatory effect to provide alternative potential to conventional chemotherapy for a variety of diseases, especially in relation to host defense mechanism. From this point of view, there is a need to improve currently commercially available vaccines in order to effectively protect animals from infection. The combined use of vaccines and immunostimulatory agents is a growing and innovative approach in adjuvant development (Gautam et al. 2008; Del Giudiceet al. 2018). So the use of this combination can improve the immune response to vaccination that can aid in improvement the immune status of the animals and increase the antibody production against different serovars of Av. paragallinarum. Consequently, our study was based on the use of P. dactylifera and M. piperita as an example for the herbal immunostimulant agents. They are characterized as heteropolysaccharides constructed on a glucan backbone. These structural features may be partly responsible for the strong immunomodulatory effects of P. dactylifera and M. piperita aqueous extracts. Polysaccharides sulphate dependent (sPS) present strong immunostimulatory activity (De-Jesus et al. 2015; Manlusoc et al. 2019). Our use of 1 and 2 mg either of P. dactylifera or M. piperta herbal crude acqueous extract as immunostimulants in combination with the commercially produced liquid paraffin polyvalent IC vaccine, developed agglutination antibody titer against each of the three tested strain antigens of Av. paragallinarum beginning from 2nd weeks post immunization and continued to increase at equivalent rates to the four antigens (W, Modesto, 0222 and local strain) reaching maximum rates at the 10th to 12th week post immunization when used alone or in corporation with liquid paraffin oil adjuvant IC vaccine. The immunomodulatory activity recorded in our results may be attributed to the polysaccharides and polyphenols, derived from date palm fruit that have potent immunomodulatory activity and stimulated both γδ T cells and myeloid cells. The limited analysis reported to date suggest that polyphenols and the polysaccharide fraction of the P. dactylifera fruit represent the immunomodulatory compounds in P. dactylifera (Baliga and Baliga 2011). The protection elucidated by P. dactylifera or/and M. piperta may be related to their antibacterial, antioxidant effect (Allen et al. 1997; Al-Turki and Abdel Magid 2004; Saddiq and Bawazir 2010; Bokhari and Perveen 2012) and immunomodulating potential (African pharmacopoeia 1985; Nickels 1996; Allen et al. 1997; Al-Turki and Abdel Magid 2004; Mekay and Blumberg 2006; Al-farsi and Lee 2008; Awaad et al. 2010; Saddiq and Bawazir 2010; Saleh 5
et al. 2011; Bokhari and Perveen 2012) by implementing both innate-cell mediated and humoral immune response in chickens.
Conclusion The present paper enumerates the plant-based immunoadjuvants which have shown remarkable immunostimulatory and protective properties. Two conclusions could be deducted from the present study: First, is the possible and promising use of Montanide™ ISA-201 in the poultry industry against Av. paragallinarum in addition to its use in the bovine vaccination program; Second, The proposed adjuvant candidates P. dactylifera and M. piperita were selected due to their promising results in their integral role in humoral immunity. These findings about plant-based adjuvants can be used to formulate other vaccine antigens. Clear evidence of augmentation of immune responses in vivo is to be further fractionated to identify the most active adjuvant components.
6
References African pharmacopoeia (1985) Organization of African Unity, Scientific Technical & Research Commission, Lagos. Vol. 1, first ed. Al Farsi MA, Lee CY (2008) Nutritional and functional properties of dates: a review. Crit Rev Food Sci Nutr 48:877–887. Allen VM, Fernandez F, Hinton MH (1997) Evaluation of the influence of supplementing the diet with mannose or palm kernel meal on Salmonella colonisation in poultry. Br Poult Sci 38:485-488. Al-Turki AI, Abdel Magid HM (2004) Bacterial Contamination of Date Fruits During Postharvest Handling. Pak J Biol Sci 7:611-614. Awaad MHH, Abdel-Alim GA, Sayed KSS, Ahmed KA, Nada AA, Alkhalaf AN (2010) Immunostimulant effects of essential oils of peppermint and eucalyptus in chickens. Pak Vet J 30:61-66. Baliga MS, Baliga BRV, Kandathil SM, Bhat HP, Vayalil PK (2011) A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Res Int 44:1812-1822. Brash ML, Charlton BR, Fitz-Coy SH, Fulton RM, Julian RJ, Jackwood MW, Ojkic D, Newman LJ, Sander JE, Shivaprasad HL, Wallner-Pendleton E, Woolcock PR (2013) Avian Disease Manual 7th Efition American Association of Avian Pathologists Kennets Square, Pennsylvania, USA. 300 pages. Bokhari NA, Perveen K (2012) In vitro inhibition potential of Phoenix dactylifera L. extracts on the growth of pathogenic fungi. J Med Plants Res 6:1083-1088. Boucher CE, Theron CW, Jansen AC, Bragg RR (2014) Transcriptional profiling of chicken immunity-related genes during infection with Avibacterium paragallinarum. Vet Immunol Immunopathol 158:135-142. CABI, current year (2019) infectious coryza In: Invasive Species Compendium. Wallingford, UK: CAB International. www.cabi.org/isc. Catanzaro M, Corsini E, Rosini M, Racchi M, Lanni C (2018) Immunomodulators Inspired by Nature: A Review on Curcumin and Echinacea. Molecules 23: 2778. Dar P, Kalaivanan R, Sied N, Mamo B, Kishore S, Suryanarayana VV, Kondabattula G (2013) Montanide ISA™ 201 adjuvanted FMD vaccine induces improved immune responses and protection in cattle. Vaccine 31:3327-3332. De Jesus Raposo MF, de Morais AMB, de Morais RMSC (2015) Marine Polysaccharides from Algae with Potential Biomedical Applications. Marine Drugs 13:2967–3028. 7
Del Giudice G, Rappuoli R, Didierlaurent AM (2018) Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Seminars in Immunology 39: 14-21. Gautam M, Gairola S, Jadhav S, Patwardhan B (2008) Ethnopharmacology in vaccine adjuvant discovery. Vaccine 26:5239-40. Gong Y, Zhang P, Wang H, Zhu W, Sun H, He Y, Shao Q, Blackall PJ (2014) Safety and efficacy studies on trivalent inactivated vaccines against infectious coryza. Vet Immunol Immunopathol 158:3-7. Kharissova OV, Kharisov BI, Oliva González CM, Méndez YP, López I (2019) Greener synthesis of chemical compounds and materials. R. Soc. open sci. 6: 191378. Kaurav M, Madan J, Sudheesh MS, Pandey RS (2018) Combined adjuvant-delivery system for new generation vaccine antigens: alliance has its own advantage. Artificial Cells, Nanomedicine, and Biotechnology 46:sup3, S818-S831 Manlusoc JKT, Hsieh CL, Hsieh CY, Salac ESN, Lee YT, Tsai PW (2019) Pharmacologic Application Potentials of Sulfated Polysaccharide from Marine Algae. Polymers 11: 1163. Mastelic B, Ahmed S, Egan WM, Del Giudice G, Golding H, Gust I, Neels P, Reed SG, Sheets RL, Siegrist CA, Lambert PH (2010) Mode of action of adjuvants: Implications for vaccine safety and design. Biologicals 38:594-601. Mekay DL, Blumberg JB (2006) A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L). Phytother Res 20:619-633. Nickels CHF (1996) Antioxidants improve cattle immunity following stress. Anim Feed Sci Tech 62:59-68. Ruiz SM, Bendezu J, Guevara RC, Montesinos R, Requena D, Moreau LC, Ávalos AM, Fernández-Díaz M (2018) Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza. BMC Vet Res 14: 411. Saddiq AA, Bawazir AE (2010) Antimicrobial activity of date palm (Phoenix dactylifera) pits extract and its role in reducing the side effects of methyl prednisolone on some neurotransmitter content in the brain, hormone testosterone and adulthood. ISHS Acta Hortic. IV International Date Palm Conference, p. 882. Sakamoto R, Sakai AT, Ushijima T, Imamura T, Kino Y, Honda T, Sakaguchi M (2012) Development of an Enzyme-Linked Immunosorbent Assay for the Measurement of Antibodies Against Infectious Coryza Vaccine. Avian Dis 7:e42-e43.
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Author statement Kamelia M. Osman: Conceptualization, Supervision, Writing, Reviewing and Editing Osama E Kamal and Marwa M Ahmed: Data curation, Methodology and Writing original draft preparation Heba N. Deif: Writing original draft
Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Phoenix dactylifera, Mentha piperita and MontanideTM ISA-201 as Immunological Adjuvants in a chicken model Kamelia M. Osman ConceptualizationSupervisionWritingReviewing and Editing , Osama E Kamal Data curationMethodology and Writing original draft preparation , Heba N. Deif Data curationMethodology and Writing original draft preparation , Marwa M Ahmed Writing original draft PII: DOI: Reference:
S0001-706X(19)31447-0 https://doi.org/10.1016/j.actatropica.2019.105281 ACTROP 105281
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
18 October 2019 20 November 2019 20 November 2019
Please cite this article as: Kamelia M. Osman ConceptualizationSupervisionWritingReviewing and Editing , Osama E Kamal Data curationMethodology and Writing original draft preparation , Heba N. Deif Data curationMethodology and Writing original draft preparation , Marwa M Ahmed Writing original draft , Phoenix dactylifera, Mentha piperita and MontanideTM ISA-201 as Immunological Adjuvants in a chicken model, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105281
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Phoenix dactylifera, Mentha piperita and Montanide™ ISA-201 as Immunological Adjuvants in a chicken model
Kamelia M. Osman 1*, Osama E Kamal3, Heba N. Deif 1, Marwa M Ahmed3
1*
Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt
2
Department of Community Health, National University, San Diego, CA USA
3
Veterinary Serum and Vaccine Research Institute, Ministry of Agriculture, Egypt
*Corresponding author, e-mail: [email protected]
1
Abstract This study evaluated plant-based immune-adjuvants from crude extracts of Phoenix dactylifera and Mentha piperita as promising adjuvants for vaccines because of the limited side effects associated with plant extracts. In addition, Montanide™ ISA 201 previously used in vaccines in cattle. Eight different infectious coryza (IC) vaccines were prepared from three serovars [A (W strain and local strain), C (Modesto strain) and B (0222 strain)] with eight Avibacterium. paragallinarum vaccines adjuvants formulae using liquid paraffin, Montanide™ ISA 71, Montanide™ ISA 201, and Montanide™ Gel adjuvants, P. dactylifera and M. piperita as immune-stimulants at a concentration of 1 mg and 2 mg incorporated with or without liquid paraffin oil as an adjuvant. These vaccines were applied in a chicken model. After a single immunization, the eight vaccine formulations were evaluated using the ELISA and Microplate agglutination test. Evidence of protection in the immunized birds was based on the results after challenge and bacterial isolation. The incorporation of the crude aqueous extract of P. dactylifera or M. piperita at a concentration of 2 mg in a liquid paraffin oil adjuvanted IC vaccine could be employed as an efficient adjuvant for chicken to IC vaccine to enhance immune responses. Also,Montanide™ ISA 201 may be the best adjuvant to be used to enhance the protective response against Av. paragallinarum. Our results confirm that aqueous extracts of M. piperita leaves and P. dactylifera fruit have immunomodulatory potentials in vivo and elevated serum antibodies against Av. Paragallinarum. Keywords: P. dactylifera and M. piperita extracts adjuvants; Av. Paragallinarum; Montanide™ ISA-201; ISA-71 and Montanide™ Gel
2
Montanide™ is a family of oil-based adjuvants that have been used in trial vaccines in human and animals (Dar et al. 2013). To date, the adjuvant potential of ISA-201 for Avibacterium paragallinarum poultry vaccine, has not been previously reported. Immunostimulators are substances (e.g. plant extracts) that stimulate the immune system by inducing activation or increasing activity of any of its components (Catanzaro et al. 2018). The most important advantage of plant extracts is that they have minimal adverse side-effects, since they are biodegradable compounds (Kharissovaet al. 2019). Additionally, they are easy to extract, relatively safe, inexpensive, often available (Mastelic et al. 2010). Avian infectious coryza (IC) is an acute upper respiratory tract infection in chickens that is caused by the Gram-negative bacterium Av. Paragallinarum (Ruiz et al., 2018). It is believed to be evolving as a re-emerging worldwide disease of poultry that affects all ages of chickens of both indigenous and laying hens in poultry farms(Gallus gallus domesticus), in pheasants (Phasianus) and guinea fowls (Numida) and Japanese quails (Coturnix japonica) (Brash et al. 2013; Thenmozhi and Malmarugan 2013; CABI,, 2019). Therefore, the present study was undertaken to compare the serological responses and protective efficacy of an inactivated trivalent IC vaccine formulated with a local strain and two international strains of Av. paragallinarum in Montanide™ ISA-201, ISA-71 and Montanide™ Gel in poultry. Also, this study illustrates the enhancing effect of the aqueous extracts P. dactylifera or M. piperita as immunostimulants for IC vaccines. A total of 550 eight-week old SPF white leghorn birds were used. Local field strain according to Page serovar A was originally isolated from an outbreak of IC in a laying flock in Egypt, confirmed at the species level and serotyped using a Page scheme of serotyping with antisera against reference serovars (Blackall et al. 1990). The reference strain 0222 (Page serovar B) was obtained from Dr. R. B. Rimler, USDA National. Animal Disease Center, Ames, Iowa, USA. The reference strains W (page serovar A) and Modesto (Page serovar C) were obtained from MSD Animal Health/Intervet International bv., Boxmeer, The Netherlands. Preparation of crude aqueous extracts of P. dactylifera (date or date palm) and M. piperita (peppermint) leaves were prepared in cold distilled water (1:3 ratio, weight to volume) and kept for 48 hours at a temperature of
with continuous stirring The extracts of both P.
dactylifera and M. piperita were freeze-dried to give a yield of 12.47 g. This was reconstituted separately in distilled water to give the required doses of 1 or 2 mg/kg body weight of the extract used in this study. Av. paragallinarum strains [(Av. paragallinarum references strains (W strain of serovar A, Modesto strain serovar C and 0222 strain of serovar B) as well as a local strain of Av. paragallinarum (serovar A) were inoculated into cBHI medium for 24 h at 37°C. The strains were inactivated with 0.25% formalin, then washed with normal saline. The bacterin of each 3
strain of Av. paragallinarum was adjusted to 6.0× 108cfu/ ml of serovar A (W strain), 1.0 ×109cfu/ml of serovar B (0222 strain) and 8.0 × 108cfu/ ml of serovar C (Modesto strain) as well as 6.0 ×108cfu/ ml of local strain (serovar A). Experimental polyvalent vaccine was adjuvenated with various adjuvants as outlined in Tables S1 and S2. The procedures of vaccine emulsification were carried out according to the SEPPIC vaccine department laboratory, France (www.seppic.com). At 10 weeks of age, the chickens were inoculated using one experimental vaccine formula for each group using the same vaccine code (V-1; V-2; V-3, V-4, V- 5, V- 6, V-7 and V-8). As a preliminary step, all birds were monitored for antibody titres against Av. paragallinarum by the Microplate agglutination test (MAT) (Wambura 2010) and Enzyme-linked immunosorbent assay (ELISA) (Sakamoto et al. 2012). At week 3 post immunization, each bird was challenged once by topical administration into the infra-orbital eye area, consisting of 100 μl (~108 CFU) of the bacterial broth reconstituted in PBS, pH 7.0 (Gong et al. 2013). All chickens were kept under observation for 10 days post challenge. The clinical signs in the birds were recorded during the course of the experiment and scored daily in numerical values relative to the severity of the clinical signs observed (Boucher et al. 2014). The variations within and between groups were analyzed using a One-Way ANOVA followed by Tukey-Kramer multiple comparison test. Minitab 17 Statistical Software (State College, PA: Minitab, Inc.) was used for all statistical analysis. Generally, all chickens in the four groups (V-1, V-2, V-3 and V-4) immunized with the four vaccine formulations developed MAT and ELISA antibodies against each of the four tested strains of Av. Paragallinarum (Tables S1 and S2) which continued to increase at equivalent rates post-immunization, to gradually decline to reach their lowest levels by the 24th week The Montanide™ ISA 201- adjuvant vaccine formulation generated a significantly (P < 0.05) higher rate of Ab titres. The data clearly indicated that using of 2 mg of either P. dactylifera or M. piperita included in the vaccine induced higher levels of antibody titers than when they were used at a concentration of 1 mg. It should also be emphasized that, when the P. dactylifera or M. piperita were used at a concentration of either 1 mg or 2 mg and included in the liquid paraffin oil adjuvant IC vaccine as immunostimulants, they resulted in a better immune response than when the liquid paraffin oil IC vaccine was used alone. Statistically, the incorporation of either 2 mg P. dactylifera or M. piperita with liquid paraffin oil adjuvant IC vaccine, generated a significant (P<0.05) higher rate of antibody titers against the four antigens of Av. paragallinarum than using vaccine with 1 mg of each immunostimulant or the vaccine without the immunostimulants. The protective efficacy of the eight vaccine formulations after challenges are summarized in Table S3. Vaccine emulsification in adjuvants, which potentiate immune responses, are a target area of vaccine development to improve vaccine efficacy (Kaurav 4
et al. 2018). The results suggest the V-3 vaccine formulation which contain Montanide™ ISA 201 could be the best adjuvant to enhance the protective response of the candidate vaccines against Av. paragallinarum. Also, the use of immunostimulators (such as plant extract) with a vaccine can stimulate the immune system by inducing activation or increasing activity of any of its components (Mastelic et al. 2010; Yang et al.. 2018). Medicinal plants which are used as immunomodulatory effect to provide alternative potential to conventional chemotherapy for a variety of diseases, especially in relation to host defense mechanism. From this point of view, there is a need to improve currently commercially available vaccines in order to effectively protect animals from infection. The combined use of vaccines and immunostimulatory agents is a growing and innovative approach in adjuvant development (Gautam et al. 2008; Del Giudiceet al. 2018). So the use of this combination can improve the immune response to vaccination that can aid in improvement the immune status of the animals and increase the antibody production against different serovars of Av. paragallinarum. Consequently, our study was based on the use of P. dactylifera and M. piperita as an example for the herbal immunostimulant agents. They are characterized as heteropolysaccharides constructed on a glucan backbone. These structural features may be partly responsible for the strong immunomodulatory effects of P. dactylifera and M. piperita aqueous extracts. Polysaccharides sulphate dependent (sPS) present strong immunostimulatory activity (De-Jesus et al. 2015; Manlusoc et al. 2019). Our use of 1 and 2 mg either of P. dactylifera or M. piperta herbal crude acqueous extract as immunostimulants in combination with the commercially produced liquid paraffin polyvalent IC vaccine, developed agglutination antibody titer against each of the three tested strain antigens of Av. paragallinarum beginning from 2nd weeks post immunization and continued to increase at equivalent rates to the four antigens (W, Modesto, 0222 and local strain) reaching maximum rates at the 10th to 12th week post immunization when used alone or in corporation with liquid paraffin oil adjuvant IC vaccine. The immunomodulatory activity recorded in our results may be attributed to the polysaccharides and polyphenols, derived from date palm fruit that have potent immunomodulatory activity and stimulated both γδ T cells and myeloid cells. The limited analysis reported to date suggest that polyphenols and the polysaccharide fraction of the P. dactylifera fruit represent the immunomodulatory compounds in P. dactylifera (Baliga and Baliga 2011). The protection elucidated by P. dactylifera or/and M. piperta may be related to their antibacterial, antioxidant effect (Allen et al. 1997; Al-Turki and Abdel Magid 2004; Saddiq and Bawazir 2010; Bokhari and Perveen 2012) and immunomodulating potential (African pharmacopoeia 1985; Nickels 1996; Allen et al. 1997; Al-Turki and Abdel Magid 2004; Mekay and Blumberg 2006; Al-farsi and Lee 2008; Awaad et al. 2010; Saddiq and Bawazir 2010; Saleh 5
et al. 2011; Bokhari and Perveen 2012) by implementing both innate-cell mediated and humoral immune response in chickens.
Conclusion The present paper enumerates the plant-based immunoadjuvants which have shown remarkable immunostimulatory and protective properties. Two conclusions could be deducted from the present study: First, is the possible and promising use of Montanide™ ISA-201 in the poultry industry against Av. paragallinarum in addition to its use in the bovine vaccination program; Second, The proposed adjuvant candidates P. dactylifera and M. piperita were selected due to their promising results in their integral role in humoral immunity. These findings about plant-based adjuvants can be used to formulate other vaccine antigens. Clear evidence of augmentation of immune responses in vivo is to be further fractionated to identify the most active adjuvant components.
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Author statement Kamelia M. Osman: Conceptualization, Supervision, Writing, Reviewing and Editing Osama E Kamal and Marwa M Ahmed: Data curation, Methodology and Writing original draft preparation Heba N. Deif: Writing original draft
Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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