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(LC50) of Pathogenic Microorganisms and Their Antigens in Vaccine Development Saganuwan Alhaji Saganuwan*

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(LC50) of Pathogenic Microorganisms and Their Antigens in Vaccine Development Saganuwan Alhaji Saganuwan* Saganuwan BMC Res Notes (2020) 13:289 https://doi.org/10.1186/s13104-020-05126-x BMC Research Notes RESEARCH NOTE Open Access Application of median lethal concentration (LC50) of pathogenic microorganisms and their antigens in vaccine development Saganuwan Alhaji Saganuwan* Abstract Objective: Lack of ideal mathematical models to qualify and quantify both pathogenicity, and virulence is a dreadful setback in development of new antimicrobials and vaccines against resistance pathogenic microorganisms. Hence, the modifed arithmetical formula of Reed and Muench has been integrated with other formulas and used to deter- mine bacterial colony forming unit/viral concentration, virulence and immunogenicity. Results: Microorganisms’ antigens tested are Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aerugi- nosa in mice and rat, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas veronii in fsh, New Castle Disease virus in chicken, Sheep Pox virus, Foot-and-Mouth Disease virus and Hepatitis A virus in vitro, respectively. The LC 50s for the pathogens using diferent routes of administrations are 1.93 103(sheep poxvirus) and 1.75 1010 for Staphylococcus × × aureus (ATCC29213) in rat, respectively. Titer index (TI) equals N log10 LC50 and provides protection against lethal dose in graded fashion which translates to protection index. N is the number of vaccine dose that could neutralize the LC50. 3 11 Hence, parasite inoculum of 10 to 10 may be used as basis for determination of LC50 and median bacterial concen- trations (BC50).Pathogenic dose for immune stimulation should be sought at concentration about LC 10. Keywords: Vaccine, Pathogenicity, Model, Arithmetic, Development, Colony forming unit Introduction numbers of colony forming units of some pathogenic Many countries have renewed efort towards develop- bacteria, viruses and their antigens were determined, ment of vaccine against a number of infectious diseases, using median lethal concentrations (LC 50s) established such as mastitis caused by Staphylococcus aureus in in laboratories, with intent to calculating immunogenic bovine and human [1]. Capsular polysaccharide, virulent doses of various infectious agents. antigens [2, 3] using adhesive proteins [4] as immuno- genic derivatives, deoxyribonucleic acid (DNA), autoly- Main text sin and protein-binding polysaccharides are also used to Methods stimulate immune system [5–7]. However, Saganuwan Reference was made to journal articles on development reported toxicological basis of antidote [8] and a number of vaccines against methicillin resistance Staphylococcus of vaccines presently being developed is based on modi- aureus and other pathogenic microorganisms that cause fed arithmetical method of Reed and Muench [9]. Hence diseases in human and animals. Median lethal concen- trations (LC50s) of Staphylococcus aureus, Streptococ- cus pneumoniae, Pseudomonas aeruginosa in mice and *Correspondence: [email protected] rat, Edwardsiella ictaluri, Aeromonas hydrophila and Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Federal University of Agriculture, P.M.B. 2373, Aeromonas veronii in catfsh, New Zealand rabbit, fsh Makurdi, Benue, Nigeria and mice were translated to colony forming units. LC 50 © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Saganuwan BMC Res Notes (2020) 13:289 Page 2 of 6 of in vitro cell cultures of hepatitis A virus and Foot and 5 NcxDf LC50 = Mouth Disease virus were translated to LC1, whereas N efective dose ffty (ED-50) for Newcastle Disease vaccines was translated to ED1 -in chickens [5–20]. Te method viii. Median bactericidal concentration (MC50) formula of Reed and Muench [21] as modifed by Saganuwan [9] is determined as follows N N0 c = −BC was used for LC50 determination in various laboratories. 1+er(x 50) whereas N = Number of colonies for Protection index (PI) is equal to titration index = Nlog10 each plate. LD50, whereas N is number of titration using vaccine. ix. In vivo LD value can be replaced by tissue culture LD 50 50 No BC = (TCL50). 50 2 Derivation of LD formula 50 Tus 2BC could replace MBC 50 1 BC = BC + lc(No− ) x. 1 50 r whereas r tangent slope i. Modifed formula of Reed and Muench = on infexion LD MLD+MSD No could estimate the bactericidal intensity [23] 50 = 2 whereas MLD Median lethal = xi. Since the rate of bacterial load depends on the con- dose; MSD = median survival dose [9]. centration of neutrophils. Exponent = (− kp + g) t, where k is the second-order rate constant for Derivation of LC50 formula bacterial killing, p neutrophil concentration; Conc. initial concentration of colony forming unit per = = g frst-order rate constant for bacterial growth; ml of sample x = = t time. When concentration is double fold, triple fold and = K 2 10−8 ml per neutrophil per min; g 8 10−3 tetra fold, they are represented as 2 x X, 3 x X and 4 x X, = × = × min respectively. P > g xii. When k = critical neutrophil concentration x+2x+3x+4x LC50 = x5 ii. Hence, 10 5 Te critical neutrophil concentration = 3–4 × 10 per ml, a value of 5 105 predisposes human to bacterial ≤ × infection [24]. All of the above formulas could be applied 10x in determination of lethal concentration of immunogenic LC50 = x5 10 and anti-immunogenic agents in various models of vac- cine development. iii. LC50 = X × 5 x = initial concentration = colony forming unit; whereas Results LC50 median lethal concentration that can kill = Te colony forming unit, LC 1-, median lethal concentra- 50% of test animals; x = initial concentration; mul- tion for each pathogenic microorganism, antigen, vac- tiplication factors for initial concentration = 10 cine, animal model and their routes of administrations iv. are presented in Table 1. Te most virulent microorgan- 10 LC50 ism is Sheep Pox virus with LC50 value of 1.93 10 cfu/ x = ×4 5 ml followed by Edwardsiella ictaluri (2.8 × 10 cfu/ml), Streptococcus pneumonia(104–107 cfu/ml) and Staph- v. Number of colony forming unit (NCFU) per unit ylococcus being the least virulent in rat with IC 50 of of sample [22] 10 1.75 × 10 cfu/ml, using intradermal, intraperitoneal, intravenous and intraperitoneal route of administra- NCFU = Nc × Df. tion, respectively. Sheep was most susceptible, followed by catfsh, mice and rat being the least susceptible in the Nc = Number of colonies; Df = Dilution factor of the present study (Table 1). plate counted CFU = NcxDf vi. Terefore N Discussion Substitute x for CFU in equation v Te median lethal concentration (1.1 108 CFU) for LC50 NcxDf × ∴ = 5 N plasmid cloned neomycin (PC1 = Neo) and plasmid LC50 N 5(Nc Df) cloned neomycin methicillin resistance Staphylococ- × = × 7 vii. cus aureus (PCl-Neo-MeccA) and 1 × 10 CFU for S. Saganuwan BMC Res Notes (2020) 13:289 Page 3 of 6 Table 1 The estimated colony forming unit and median lethal concentration (LD50) of pathogenic microorganisms’ antigens and vaccines Pathogenic Animal model Antigen(s)/Strain Route CFU (LC1) LC50 cfu/ml Comments Reference(s) microbes Staphylococcus Mice pC1-Neo Intraperitoneal 2.2 107 1.1 108 Less virulent [5] aureus pC1-Neo-MeccA × × Staphylococcus Mice Fibrinogen Intravenous 2 106 1 107* Less virulent [6] aureus Fibronectin × × Staphylococcus Mice Endotoxin-free PBS Intraperitoneal 1 108 5 108* Less virulent [10] aureus × × New Castle disease Chicken Lasota vaccine Oral 0.84–1.92 4.2–9.6/ml Very virulent [11] virus New Castle disease Chicken 12 vaccine Oral 1.14–1.92 5.7–9.6/ml Very virulent [11] virus Hepatitis A Virus In vitro HAV AM75/18F In vitro 2.8 106 1.4 107 Less virulent [12] × × Streptococcus pneu- Mice Pneumococcal sur- Subcut 9 104–106 4.5 104–106 Moderately virulent [13] moniae face protein A × × Streptococcus pneu- Mice PSPA1 and 2 bound Intravenous 2 103–2 106 104–107 Moderately virulent [14] moniae to Vi polysac- × × charide Foot and Mouth In vitro cell line Serotype A, 0 and Cell culture 2.8 108 1.4 109 Less virulent [15] Disease virus (hamster kidney SAT-2 × × 21 cell line) Staphylococcus Rat Strain (ATCC29213) Intraperitoneal 3.5 109 1.75 1010 Less virulent [16] aureus × × Pseudomonas Rat (ATCC27853) strain Intraperitoneal 6 107 3.0 108 Less virulent [16] aeruginosa × × Sheep pox virus Sheep SPPV strain (Hd Intradermal 3.86 102 1.93 103 Highly virulent [17] 2012) × × Edwardsiella ictaluri Catfsh Suspension of E. Intraperitoneal 5.6 103 2.8 104 Moderately virulent [18] ictaluri × × Aeromonas New Zealand rabbit, Glycoprotein Intradermal 1 109 5 109 Less virulent [19] hydrophila fsh based-vaccine × × 8 9 1 Aeromonas veronii Fish mice Bacteriovorax strain Oral 7.2 10 > 10 PFUg− Less virulent [20] × H2 CFU colony forming unit * sublethal dose; highly virulent statistically signifcant in relation to CFU/viral concentration; moderately virulent-statistically signifcant in relation to CFU/viral = = concentrations; Less virulent statistically not signifcant in relation to CFU/viral concentrations = aureus fbrinogen in mice show that the microorgan- ml), C30 strain (1.1–22/ml) and Villegas-Glisson Uni- ism is less virulent [5].
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