CS267949B1 - Inactivated vaccine against salmonellosis of calves - Google Patents

Abstract

Rosonia refers to inactivated vaccine substance useful in immunoprofylacil salmonellosis calves that raises high degree in vaccinated animals fuzzy bifurcation. Contains bacterially 5 strains of Salmonella typhinnjrium noncompatible factors of pathogenicity, ohemioky inactivated and leveled in the same volume ratio. After triple intramuscular apláoii causes significant State of Speoifiokej Breach Against with β virulent strain of this kind of microorganism. Naohádza use in glandular and bulk to reduce economic losses morbidity and mortality of calves because of this infectious disease.

Description

The invention relates to an inactivated vaccine prepared from five selected strains of S. typhimurium, which have defined pathogenicity factors which induce a high degree of specific protection in vaccinated calves and which can be used in the immunoprophylaxis of salmonella salmonella.

Current knowledge about the influence of pathogenicity factors in aalmonel infections leads to a more critical reassessment of the areas of technological preparation of specific immunogens. Knowledge of the expression conditions of pathogenicity factors in vivo in the etiopathogenesis of salmonella infection! enabled the in vitro selection of strains suitable for the technological realization of specific immunogens capable of inducing a high degree of macroorganism defense.

Occurrence of salmonella infection! It has a growing tendency and becomes one of the infections, which significantly impairs the economic efficiency of calf rearing, but this problem is all the more serious because this infokia is transmissible to humans. The possibility of managing this disease with ohemotherapeutic agents and antibiotic agents is currently limited by the occurrence of high percentages of polyresistant strains.

Consider the findings on the effects of salmonellosis on hygiene and / or that prophylactic treatment with antibiotics! on farms does not affect the elimination of salmonella (Wray, C., Tood, JN, Hinton, M ,, 1987, Vet, Reo, 26, 293-296), Prof, BA, Shenderov from the National Antibiotic Institute of the USSR to V, Czechoslovakia, congress on Infectious and parasitic diseases (Piošlany, 22, - 24, June 1988) on the effect of antibiotics on facultatively intraoellular pathogens such as S, typhimurium replied that in an experimental model they found that antibiotics almost stimulated salmonella growth in the body as if they inhibited ioh. the point is that salmonella is detected in a fat environment even after documented cleaning and disinfection! (Wray 1975, Vet. Bul. 50, 543-548), which points to bacillocarriers. Therefore, pups at the age of 14 days are concentrated in the calf onemoone on the basis of salmonellosis not at night, but after incubation for 7 to 4 days.

In addition to environmental hygiene, age sensitivity is important in the occurrence of salmonella infections. In S. dublin the average time of onset of infection is 4 weeks and in S, typhimurium 3 weeks (Maft, 1983 - / Perceived Salmonellosis, 1983, Animal Summaries). The explanation is that the infection caused by S. dublin is relatively lateral in adult animals (Vray, CN, Todd, M., Hinton; Epidemiology of Salmonella typhimurium infection in oalvos: Excretion of S, typhimurium in the fooes of calves in different management systems, Vet.Roc., September 26, 1987, 293-296). Therefore, we consider the main to induce immunity in calves by the 18th day of their vocation, to prevent the outbreak.infection! after their companion to the toilets.

In the field of specific prophylaxis of this disease, various types of vaccines have been used on a global scale, which used the various principles of immune mechanisms in the body. Prinoipally It is possible to categorize these biopreparations into the field of nutrient, suboollular and whole cell vaccinia (inactivated),

Live vaccines contain strains that are dependent on aromatic acids or streptomycin or galactose or are harsh mutants. If experimentally found, this type of vacoin induces severe post-vaccination rococa in immunized animals, clinically similar to its own infection. In addition to the above, a serious problem from the epizootiological point of view is the excretion of vaccine strains in féoes and possible horizontal transfer of plasmids of pathogenicity and reziatenoie (ílikula, I ,, Pilipčineo, E., Le Van Tao, Holoda, E, - Immunogenicity testing of salmonella vaccines, Partial Final Report , 1987 »Pilipčineo, E, - Properties of Salmonella typhimurium strains in relation to their virulence and immunogenicity, Candidate dissertation 1986} Clarke, RC, Gyles, C, L. - Galactose epimeraeeless mutants of Salmonella typhimurium as live vaccine for calves. J.Vet.Res. 50, 1986, pp. L65-173l Baljer, G. - Salmonelloso, Tierarztliohe Umsohau Sonderdruck 39, 1984, Seite 368-375 | Mikula, I., Pilipčineo, E., Holoda, E., Topical trends of applications of Salmonella Vaccines in farm

CS 26? 94? Bl animals, 1987, Proceedings of International Symposium VAC Agrokombinát - Slušovice),

An attempt to eliminate the above-mentioned disadvantages of nutrient vaccines is the preparation of cutting suboellular substances from the microorganism Salmonella typhimurium, which have been proven to be potentially immunogens in this infection. Thus, they have been experimentally tested as vacoins against ealtdonel infection of calf ribosomes, cell wall lipopolysaccharides, boivin endotoxin and porins. The transfer of the preparation of these substances into the technological process was limited by the high technical complexity, non-reproducibility of the obtained yields and low immunogenicity.

Another trend in solving this problem has been the effort to prepare various types of inactivated adjuvant whole cell vaccines. In this vaccine, the bacterial mass has been physically inactivated; heat as well as various chemicals; formaldehyde, phenol, acetone. Subsequently, inactivated bacterial cells were adsorbed on various types of mineral carriers or emulsified in oil adjuvants. This fact has not been comprehensively accepted in this type of vaccine preparation so far, and therefore the efficacy of the vaccinates prepared so far was variable and did not guarantee solid immunity in vaccinated calves.

From this, there is a current need to address the problem of calf salmonellosis by introducing an effective vacoin that would induce a high degree of specific disease in vaccinees. At the same time, the vaccine must be designed to respect current knowledge in the field of microbial genetics and immunopetology.

The basis of our experiments proved that it is possible to produce an inactivated vaccine against salmonellosis of calves from a bacterial culture prepared by culturing strains S, typhimurium, which possesses pathogenicity factors (see Table 1),

Strains were isolated from calves affected by severe clinical ulcers with salmonellosis. The mentioned strains were stored in the Czechoslovak Collection of Microorganisms of the University J, E, Purkynč, 662 43 Brno, cl. Defenders of Peace 10. The tribes were assigned the following deposit numbers; CCM 4015, CCM 4O16, CCM 4017, CCM 4018, CCM 4019.

The authors found experimentally that optimal growth and the desired phenotypic expression are achieved using a combination of Soy nutrient medium (Bioveta n, p, - Nitra), which is a solution of amino acids obtained by enzymatic hydrolysis of delipinized soy juice and yeast extract (Bioveta n. P, - Nitra), which is obtained by thermal hydrolysis of spent yeast.

The procedure presented by us is based on the knowledge that each microorganism within a population shows variability in physiological and biological properties. This allows him to survive the sometimes seemingly lethal external influences of the environment. The pathogen is in the frame! genus in its pathogenic properties and not the individual field insulators, our results showed variability in the properties of the strains in phase variation in two planes;

a) the specified property is or will not be manifested,

b) the mentioned property is manifested minimally or maximally (it is a calculation per cell, eg, production of fimbril, toxin, seroresistance and invasiveness, adhesion as well as penetration properties - Use of knowledge about the factor pathogenicity of salmonella in the selection of vaccination strains VI-5-13 / 02, November 1985 /).

Based on our experience with faculty intraoellular pathogens in inducing immunity, the cells of the immune system are activated and antigens that realize adhesin and subsequent colonization of the gastrointestinal tract by microorganisms, as well as penetration of the pathogen, intracellular epithelial cells and parietal invasive cells. The listed properties in the selection of strains are presented in Table no. 1.

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The finding that bacterial strains differ in the production of pathogenicity factors qualitatively and quantitatively and that this dependence is also conditioned by cultivation in the appropriate nutrient medium (Mikula et al., Final Report of the SPZV, VI-5-1J / O2, November 1985) is considered important step for obtaining the immunogen. The results presented by us (Table δ, i) indicate the properties of the strain after culturing it for 10 hours at 37 ° C in soy medium enriched with 2.5 volumes of yeast extract. By administering the first dose within 3 days after delivery, the authors have found that the immunization schedule allows the induction of a degree of specific protection of vaccinees who are able to withstand experimentally oral infection with fully virulent S. typhimurium strains. When applying vacoins, our knowledge is used to test the immune system of calves. It is found that in calves 24 hours after sine birth, they recorded significant phagocytic activity of leukooytes and an increase in the proliferative activity of lymphocytes in the first week of life. This indicates the suitability of the application of the immunogen in the first child after birth, 0 analogous results reported at the Immunological Congress in 1988 in Pilsen Toman, Krejčí, Menžik, Štěpánek: Immunocompetence of newborn calves and the possibility of its influence. Immunological newsletter δ. 1/88, etr. 69,

The inactivated vaccine according to the invention contains 5 to 8x10 ¹ θ of bacterial bodies of Salmonella typhimurium strains CCM 4015, CCM 4016, CCM 4017, CCM 4018, CCM 4019 in the same volume ratio, in 1 ml of physiological saline, inactivated 0.2 to 0.5 g phenol per 100 ml of bacterial suspension.

The dose of vaccine is 2 ml for each administration. The length of immunity in calves νοδί infection S, typhimurium from the point of view of the etiopathogenase of this disease is sufficient One month.

For challenge purposes, a strain was used which, after oral administration at a dose of 2 to 6x10 6 KTZ in ml, was able to induce a severe form of salmonellosis in the control group of calves with death within 12 days of infection (tab, δ, 2).

Tab. no. 1

CHARACTERISTICS OF INDIVIDUAL STEMS

Tested factors CCM 4015 CCM 4016 CCM 4017 CCM 4018 CCM 4019 Adhesion (x) onterocytes 19.60 17.74 13.29 19.24 12.17 VEKO burťky 24.26 10.75 12.20 31.56 14.06 MDBK cells 23.96 13.82 12.85 19.50 14.32 Enterotoxin production: skin test + + + - + FAITH + + + - - CHEO 4 * + + - - lig. calf hooks 4 * + + - - Seroresistenoia (£) 20.26 15.94 240.55 1.18 4 - 4.40 Sereny's test + (48 hours) - + (72 hours) - + (72 hours) Transferrin sorption (¢) 23.7 90.8 27.7 12.0 1.7 Colioin production: «· - - + - Homolysin production: + + + + +

x = pre-morbid number of adherent bacteria on the bimetry system tested

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Vaccine immunogenicity assays performed on 14 calves (Table, δ, 2) indicated that the inactivated vaccine induced 100% of the protocol. A control group of 10 calves that were immunized as placebo with soy medium survived an oral infection of 10 CFU only by day 9, and the calf codes died with typical signs of salmonella infection.

The tests used to monitor immunity in calves (phagooytic activity of leukooytes; index of phagooytic activity of leukoytes);

a) after the second immunization there was a significant increase (0.05) of phagooytic activity (tab, δ. 3) and phagooytic index (tab, δ. 4).

b) After infoculation in immunized calves, the number of E-rosette activities increased significantly! (T-lymphocytes) (p> <0.01), High significance after infection! indicates activation of T-lymphocytes even in the absence of adjuvants.

o) After 3 immunizations, the inhibition of leukocyte migration was significantly increased (0.01) (Table δ, 6), expressing the increased production of migraine-inhibited factor. This indicates that vaccination affects proliferation and induces increased lymphocyte reactivity after application of the inactivated vaccine prepared by us in in vitro testing.

The model developed and used by us for testing the immunogenic value of a vaccine substance on calves is difficult to apply from the point of view of practical implementation in the technological preparation of vacoins; from the viewpoint of obtaining suspect animals; possible transmission of LV age infection and high economic intensity. For these reasons, we used a mouse model for testing (Mlkula, I ,, Le Van Tao, Pilipéinoo, E, 1986, 40-551.

Tab, δ, 2

IMMUNIZATION OF CALVES WITH AN INACTIVATED CALL SALMONELLOSIS VACCINE

Group Inactivated vaccine substance produced Tested on teíatáoh Number of calves Infekoia S, typhlmurium 4/5 = CFU Protekoia * Average survival time I. VŠV Košice liquid Bioveta n.p. Nitra, 1 year after production 4 10 ⁶ $ 100 more than 21 days II. VŠV Košioo lyophilized Bioveta n.p. inside, 1 year after production 4 10 ⁶ $ 100 more than 21 days III. Control Bioveta n.p. Nitra 4 10 ^á 0 9 days IV, Bioveta n.p. Liquid Nitra VŠV Košice 3 mo. after production 3 10 ⁶ 10 ⁶ 100 £ more than 21 days IN. Control VŠV Košice 3 0 8.6 VI, VŠV Košice liquid VŠV Košioe 1 month after production 3 10 ⁶ 100 £ $ 100 vii. Control VŠV Košioo 3 10 ⁶ 0 9.3

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Tab.No.3

INDEX OF PHAGOCYTAR ACTIVITY OF LEUKOCYTES IN CALVES AFTER IMMUNIZATION * AND FOLLOW-UP INFECTION WITH S.TYPHIMURIUM strain 4/5

AGE OF CALVS / ON DAYS / ADVICE COLLECTION TELIAT GROUP i K - + X - s d P x - sd 2-3 i ₁ I HUN IZÁCI A '/1 DAY/ 5.5 - 0.45 0 .-5.6 - 0.85 ; 6-7 i ‘, —- 2. _____________ / immunization \ 3 \ / 3.DAY/ / 6.56 - 0.56 0 6.23 and 0.60 9-10 9.3 and 0.87 <0.01 6.37 and 0.67 13-14 4. / --------------- < / IMMUNIZATION \ \ /12.DAY/ 2 5. 9.43 and 1.29 <0.05 6.37 and 1.06 16-17 9.23 and 1.6 <0.05 6.03 - 1.00 20-21 , INFECTION ° ’/18.-19.DAY/ 10.8 and 1.72 <0.05 6.27 and 0.85 23-24 7. 12.4 and 6.7 <0.001 5.57 and 0.71 2 7-28 8. 14.3 and 1.70 <0.01 8.4 and 0.56 30-31 9. 14.8 í 1.93 ________ N ——_— + + +

LEGEND: + = DEATH: 1 BODY

K = CONTROL GROUP = 3 TOPICS n = Statistically not evaluable * IMMUNIZATION = ACTIVATED VACANT SALMONELLOSIS VACCINE

I = IMMUNIZED GROUP = 3 CALVES

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T ab. no. 4

PHAGOCYTAL ACTIVITY OF LEUKOCYTS IN CALVES AFTER IMMUNIZATION AND FOLLOW-UP INFECTION WITH S. TYPHIMURIUM STRAIN 4/5

AGE TELAÍA / IN THE DAYS / ADVICE COLLECTION TELIAT GROUP I. K x - sd P - + x - sd 2-3 1. immunization / DAY 1 / 62 - 6 0 67 - 4.04 6-7 / immunizationAciaX \ /5.DAY/ / 67.6 - 7.8 0 65.6 - 7.02 9- 1 0 85.3 - 6.11 <0.05 64.3 - 5.68 13-14 ⁴ · / ihunizAcia \ (/12.D EŇ / y 5. 85.3 - 0.08 <0.05 63.3 - 4.04 16-17 20-21 83 - 9.53 <0.05 64.6 - 5.68 6. INFECTION /18.-19.DAY/ 84.3 - 3.05 <0.01 63.6 and 5.86 23-24 7. 89.6 - 6.93 <0.01 66.3 - 5.68 27-28 8. 89.3 - 7.02 <0.05 72.6 and 4.72 30-31 9. 87.6 - 4.04 N + + +

LEGEND: + = DEATH: 1 BODY.

K = CONTROL GROUP = 3 CALVES n = Statistically invaluable * immunization = inactivated vaccine against

SALMONELOSIS OF TELIAT.

IMMUNIZED group of calves

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Tab.δ.5

E - ROSES FOR T - LYMPHOCYTES IN CALVES AFTER IHUNIZATION * AND SUBSEQUENT INFECTION WITH KHENOM S.TYPH I MURIUM 4/5

VčK TELAÍA Z V OŇOCHZ ADVICE COLLECTION I. GROUP IELIAT K “+ x - sd P - + x - sd 2-3 1. IMMUNIZATION / 1 DAY/ 20 and 2 0 22.6 - 3.79 ' 6-7 / immunization \ \ /5.DEN/ / 23.6 and 4.72 0 24 - 4.58 9-10 24.3 and 3.51 0 23.6 - 1.52 13-14 / immunization \ \ / 12.PEN/ / 25 - 2.6 0 23.3 - 3.51 16-17 28 - 5.56 0 23.6 - 3.05 20-21 6. INFECTION /18.-19.DAY/ 26.3 - 2.08 0 24 - 2.65 23-24 7. 38.6 - 2.16 <0.01 25 and 3.6 27-28 8. 37.6 - 1.52 <0.05 30.3 - 3.5

LEGEND: + = FIRE: 1 TEÍA

K = CONTROL GROUP = 3 DATES

N = Not statistically evaluable * IMMUNIZATION = INACTIVATED TEEIAT SALMONELLOSIS VACCINATE

I = IMMUNIZED GROUP = 3 CALVES

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Tab.δ.6 *

INHIBITION OF NIGR, CIE LEUKOCYTES IN CALVES AFTER IMMUNIZATION AND BLIND

INFECTION WITH THE TYPE OF S. TYPHIMURIUM 4/5

age TELIA! / V DHOCH / ADVISE the subscription group of calves I K x - sd P x - sd 2-3 1. immunization /1.PEŇ/ 0.95 - 0.06 0 0.99 Í 0 09 6- 7 2. .--------------------. / immunizationAciaX \ /5.PEŇ/ / 1.13 and 0.25 0 1.03 - 0.12 9-10 0.85 - 0.06 0 0.95 - 0.05 13-14 ⁴ · / immunizationAciaX \ / 12.PEN/ / 5. 0.82 - 0.06 0 1.00 and 0.1 16-17 0.64 - 0.07 <0.01 0.9 - 0.03 20-21 , INFECTION /18.-19.PEŇ/ 0.56 and 0.06 <0.01 0.98 and 0.07 23-24 7. 0.45 - 0.11 <0, 01 0.87 to 0.08 27-28 8. 0.48 - 0.12 <0.05 0.80 - 0.06 30-31 9. 0.52 - 0.09 N + + +

LEGEND: + DEATH: 1 TEUA κ = control group = 3 teCatA n = Statistically invaluable * immunization - inactivated vaccine against

CALVON SALMONELOSIS i = immunized group = 3 teCatA

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Example δ, 1

Salmonella typhimurium strains CCM 4015, CCM 4016, CCM 4017, CCM 4018, CCM 4019 are propagated in sterile soy medium which is enriched with a 2.5 volume solution of yeast extract. The medium is tempered to 37 ° C. Each strain of this microorganism is propagated separately. After culturing for 10 hours at 37 ° C, we tested samples of each strain to determine optical density, bacterial mass pH and sterility. The resulting density was 25 and pH 7.2.

The obtained bacterial mass was chemically inactivated as follows; To 100 g of bacterial mass we added 400 ml of physiological saline. The bacterial suspension was subsequently homogenized by gentle stirring in a water bath at 56 ° C for 1 hour and inactivated with 0.3 volume phenol for 96 hours at room temperature. The supernatant was removed by centrifugation. , resuspend the sediment with saline so that 1 ml of inactivated substance contains 7x10 ¹⁰ bacterial cells,

The inactivated vaccine was free of bacterial and fungal contamination. After subcutaneous administration of 0.3 .mu.l of vaccine, none of the 10 vaccinated mice died within 8 days.

A single intraperitoneally administered inactivated vaccine at a dose of 0.5 μl followed by 14 days by oral infection at a dose of 10 g of germ (CFU) was able to induce inhibition of the penetration of the virulent strain into the spleen.

The immunogenic value of the inactivated vaccine was determined over 12 days by the mean time to death after infection. It was zero for immunized calves compared to 44.5 for calves not immunized under laboratory conditions,

Claims (2)

OBJECT OF THE INVENTION 1. Inactivated calf salmonellosis vaccine, characterized in that it contains 5 to 8 x 10 ¹⁰ Salmonella typhimurium bacterial bodies of strains CCM 4015, CCM 4016, CCM 4017, CCM 4018, CCM 4019 in the same volume ratio, in 1 ml of inactivated saline. 0.2 to 0.5 g of phenol per 100 ml of bacterial suspension.