Comp. Immun. Microbiol. infect. Dis. Vol. 15, No. 1, pp. 31-40, 1992 Printed in Great Britain.All rights reserved
0147-9571/92$5.00+ 0.00 Copyright © 1992PergamonPress plc
AN INACTIVATED VACCINE AGAINST R I N G W O R M K. WAWRZKIEWICZ a n d J. WAWRZKIEWICZ Department of Veterinary Microbiology, Agricultural Academy, 20-033 Lublin, Akademicka 12, Poland A~tract--A new vaccine against ringworm, containing the inactivated Trichophyton verrucosum strain, was assessed on guinea pigs and calves under experimental conditions and on three herds of cattle under natural conditions. The vaccine elicited a distinct immune response of the cellular type. This type of immunity assessed by the migration inhibition test of leukocytes corresponded to the immunity evaluated by the challenge. In herds in which there were from 30 to 67% of naturally infected animals with T. verrucosum, two doses of the vaccine resulted in after 4 weeks a decrease of the number of animals with clinical changes from 40.5 to 100% depending on the group of animals under study. Key words: ringworm (trichophytosis), inactivated vaccine, prophylaxis, treatment, delayed
hypersensitivity test, migration inhibition test. Rrsum~-L'etficacit6 d'un nouveau vaccin inactiv6contre la trichophytie du brtail a 6t6 6tudire sur des cobayes et des veaux dans des conditions exprrimentales et sur 3 troupeaux de b&ail dans des conditions de contagion naturelle. Le vaccin contenant la souche Trichophyton verrucosum a entrain6 une nette rrponse immunologique du type cellulaire. La rraction immunologique, apprrcire ~i l'aide du test d'inhibition de migration du leucocytes etait en correlation avec l'immunitr,drtermin~e par epreuve infestante. Dans les troupeaux off 30 ~i670 d'animaux ont ~t~ infestrs avec la souche T. verrucosum on a constat6 aprrs 4 semaines, aprrs avoir requ deux doses de vaccin, une diminution du nombre de'animaux prrsentant des sympt6mes cliniques de maladie dans la proposition de 40,5% a 100% par rapport au temoins. Mots-clefs: trichophytie/trichophytosis, vaccin inactivr, prophylaxie, traitement, test d'hypersensibilit6 tardive, test d'inhibition de migration.
INTRODUCTION R i n g w o r m (trichophytosis) is a c o m m o n debilitating disease of animals, chiefly of cattle, which is economically i m p o r t a n t to the breeder because it entails loss o f p r o d u c t i o n , costs o f treatment, a n d a b a n o n export. A n i m a l s trichophytosis is also a n i m p o r t a n t p r o b l e m for public health due to its easy t r a n s m i s s i o n to m a n [l~t]. M o d e r n c o n t r o l of the disease in a n i m a l s consists in a d m i n i s t e r i n g different i m m u n o p r e p a r a t i o n s . U p till n o w some vaccines have been developed a n d / o r applied in veterinary practice against r i n g w o r m in cattle [5-10], in horses [11], a n d in fur a n i m a l s [12, 13]. All these p r e p a r a t i o n s are m o n o v a l e n t alive vaccines which, however, have short storage life if n o t lyophilized. I n addition, all these strains in the vaccines, like other a t t e n u a t e d strains, are d a n g e r o u s for individuals with i m m u n o l o g i c a l defects; they can also revert to virulent forms a n d therefore m a y constitute some d a n g e r o f infection for h u m a n beings. In order to avoid all these disadvantages a new sort o f vaccine has been developed by the selection o f a highly i m m u n o g e n i c strain o f T r i c h o p h y t o n v e r r u c o s u m a n d its i n a c t i v a t i o n u n d e r proper conditions. Tests o n the vaccine activity have been carried out o n guinea pigs a n d calves u n d e r strictly controlled c o n d i t i o n s a n d o n cattle u n d e r field c o n d i t i o n s to assess n o t only the prophylactic b u t also the therapeutic value o f the vaccine. 31
32
K. WAWRZKIEWICZand J. WAWRZKIEWICZ MATERIALS AND METHODS
Vaccine. A suspension of T. verrucosum (Tv) strain No. 43, selected on the basis of its antigenic structure [14], was used to prepare the vaccine according to our own method [15]. Briefly, the strain producing spores abundantly was grown in Sabouraud's liquid medium and its culture suspension containing approx. 5 x l 0 7 C F U ml ~ was inactivated with formalin (0.3%). After checking its sterility and harmlessness on mice and guinea pigs the vaccine was administered intramuscularly. Immunisation Guinea pigs. Each of 18 guinea pigs, free from mycotic dermatoses, was injected once with 0.5 ml of the vaccine. An immune response was evaluated by the allergic skin (AST) and the migration inhibition test (MIT) in 12 animals. The other 6 vaccinated individuals were exposed to the "challenge" test of virulent strains of T. verrucosum and T. mentagrophytes. Each animal was administered the virulent strains (105 C F U ml 1) by rubbing into both sides of the body. Six unvaccinated guinea pigs served as control animals. Calves aged 5 - 8 days. The first group of animals (I) comprised 4 experimental calves and 1 control; they were vaccinated in autumn at a low air temperature and low relative humidity (58 67%) in the stalls. The second group, containing 6 experimental calves and 2 control animals (II), was vaccinated in spring at a fairly high air temperature (20-22°C) and a high relative humidity (83-91%) of the surroundings. In the first days of their life some of the animals were given colostrum in the amount of 3 6 litres daily and others from 4.5 to 9 1. Calves aged 1-3 months. Studies were also performed in two seasons altogether on 15 calves of the lowland black and white breed. The first experiment was carried out on 4 calves and 2 controls in the autumn-winter period, and the second on 6 vaccinated and 3 control animals in the spring-summer period. The animals were being housed for 4 months in separate stalls, each for 2--3 animals. The vaccine was given twice, at intervals of 14 days at a dose from 5 to 8 ml depending on the animals age. Evaluation of immunity under field conditions Studies were performed on cattle in the autumn winter period under natural conditions of infection. The animals were housed together in an infected environment where trichophytosis had been recorded. The observations concerned 639 vaccinated animals of different age, some of which showed the ringworm signs confirmed by mycological examinations. Seventeen unvaccinated animals with the signs of ringworm served as controls. The lesions were found chiefly on the neck and head, but a general dissemination over the body occurred as well. For prophylactic purposes the vaccine was administered according to Aamodt et al. [16] at the dose from 5 to 8 ml, twice, 14 days apart, and in animals with mycotic lesions the doses were 50% higher. The efficacy of the preparation was assayed on the basis of lesions recession and the percentage of cured animals.
Allergic skin test (AST) A filtrate of a 4 months-old culture of the T. mentagrophytes 1 strain in Sabouraud's liquid medium with 4% of dextrose, containing 3.4 g ml ~ protein and 34.5 g ml t sugar, was used as the allergen. Guinea pigs were injected with 0.1 ml of the allergen intracutaneously in the scapula region. The results were read after 24 and 48 h taking into consideration the diameter of redness, swelling and necrosis. In the case of the cattle, the allergen was administered in the neck region at a dose of 0.4 ml and the results were
An inactivated vaccine against ringworm
33
assessed after 24h. An increase of at least 3 mm of the skin fold thickness over the prechallenge state was regarded as a positive result. Migration inhibition test ( M I T )
The test was performed in capillaries according to the method described earlier by Wawrzkiewicz and Zi6tkowska [17]. The source of sensitized cells in guinea pigs was the spleen and peripheral blood in the case of the cattle. A suspension of leukocytes was prepared after the method of Moreno-Lopez [18]. Trichophytin as a specific antigen was prepared from the T. mentagrophytes 1 strain and applied at a dose of 0.005 ml ~ of the medium. Average values of migration areas of cells were calculated on the basis of 15 cultures with the antigen and 15 cultures without the antigen. Migration inhibition was calculated from the following equation: 1 -
average migration area with antigen average migration area without antigen
x
100.
Taking into account the findings from in control animals a significant degree of cell migration inhibition was accepted to be 20% or more. Challenge test
An experimental infection of guinea pigs was done 6 weeks after vaccination for 2 consecutive days by rubbing a dense suspension of virulent strains into the depilated and scarified skin in the dorsal region. T. mentagrophytes was administered on one side and T. verrucosum on the other side of each animal. The injected guinea pigs were observed for 4 weeks and the material with pathological changes was assayed mycologically. Calves were experimentally infected 8 weeks after receiving the first dose of the vaccine. A standardized suspension of the T. verrucosum Z strain, containing from 105 to 10 3 CFU ml i, was rubbed for 3 successive days into the skin depilated and scarified on both sides of each animal. The animals were observed for 2 months and all cases of clinical lesions were confirmed by mycological examinations. Statistics
The values of the allergic skin test and migration inhibition test (Table 2) were characterized by the mean (M) and standard deviation (SD). The significance of differences between the means was checked by the Student's t-test or Cochran and Cox's test [19]. The effects of prophylactic and therapeutic action of the inactivated vaccine in infected herds with ringworm were expressed in percentages; the significance of differences between the percentages was checked by Z 2 test (Table 4). A 5% risk of conclusion (P < 0.05) was accepted. RESULTS Immune response in guinea pigs
Table 1 shows the immune response of guinea pigs estimated by the allergic skin test (AST), the migration inhibition test (MIT), and the results of challenge with virulent T. verrucosum and T. mentagrophytes strains. Out of six immunised animals assayed by the allergic skin test, five displayed a delayed type hypersensitivity producing large areas of redness, swelling and/or necrosis at the site of the allergen injection. In one vaccinated guinea pig (No. 10) no changes were noticed, apart from mild redness. In other seven animals immunised and examined by the MIT, with one exception No. 10, the spleen cells migration inhibition was significant, especially on days 14 and 21. Six guinea pigs of the
28
30
R
++
++
7 S
+ +
++
N
25
25
R
+
+
14 S
+
+
N
10~
25
R
+
21 S N
10
R
28 S N
R: redness; S: swelling; N: necrosis; x: pregnant; 1 + to 4 + : intensiveness of clinical lesions.
Controls lc 2c 3c
7 8 9 10 11 12
Guinea pig No.
Allergic test (days)
7
28,4
24.7 62.7 61.6
14
50.0 46.4
21
Migration inhibition test % of inhibition (days)
Table 1. I m m u n e response of guinea pigs vaccinated against ringworm
14.7
28
4c 5c 6c
13 14 15 16 17 18
Guinea pig No.
3+ 2+ 2+
m
E
3+ 3+ 3+
1+ 1+ 1+ I+ 1+
2+
Challenge T.v. T.m. Clinical lesions
N
N
N
e~
N
N
4.5-9.0 4.5-9.0 4.5-9.0 4.5-9.0 4.5-9.0 3.045.0 3.0-6.0 3.0~.0 3.0~.0 3.0~.0 4.5-9.0
3.ff6.0
Calf No.
2 3 4 5 6 7 8 9 10 1-5
6-10
n.t.: not tested.
Controls 11 12 13
1
Daily dose o f collostrum in litres
1.8 1.4
2.0 3.9 2.3 1.7 5.10 +0.86 2.48 ±0.98
6/4+* 11/3+ 3/2 + 17/1 + 132/35/4 + 125/3 + 67/2+ 23/1 + 120/-8/4 + 12/3+ 4/2 + 6/1 + 70/--
37:169
250:370
30:100
I 2-3 months autumn
II 2~, months autumn
II1 7 8 months winter
3/4 + 3/3 + 3/3 + 2/2 +
I1
31/4 + 132/3 + 68/2+ 18/1 + 121/-
1
4/2 + 2/1 + 94/-
14/4+ 3/3+ 2/2 + 18/1 + 132/ 17/4 + 62/3 + 120/2+ 31/1 + 140/-
2
17/4 + 62/3 + 120/2+ 31/I + 140/--
3
4
5/2 +
4/4 + 2/3 + 3/3 + 3/2 +
100/
9/2 + 13/1 + 147/4/4 + 14/3 + 19/2+ 29/1 + 304/-
Clinical status following vaccination Weeks
*Numerator: number of animals; denominator: intensiveness of clinical lesions.
III
6/4 +
1
Controls
Clinical status before vaccination
100
73.6
40.5
% of cured animals after 4 weeks
Table 4. Effects of inactivated vaccine on the course of ringworm in cattle--prophylaxis and treatment
Number of infected animals in relation to the whole herd
Group age of animals and season of observation
10.4
16.6
X2
0147-9571/92$5.00+ 0.00 Copyright © 1992PergamonPress plc
AN INACTIVATED VACCINE AGAINST R I N G W O R M K. WAWRZKIEWICZ a n d J. WAWRZKIEWICZ Department of Veterinary Microbiology, Agricultural Academy, 20-033 Lublin, Akademicka 12, Poland A~tract--A new vaccine against ringworm, containing the inactivated Trichophyton verrucosum strain, was assessed on guinea pigs and calves under experimental conditions and on three herds of cattle under natural conditions. The vaccine elicited a distinct immune response of the cellular type. This type of immunity assessed by the migration inhibition test of leukocytes corresponded to the immunity evaluated by the challenge. In herds in which there were from 30 to 67% of naturally infected animals with T. verrucosum, two doses of the vaccine resulted in after 4 weeks a decrease of the number of animals with clinical changes from 40.5 to 100% depending on the group of animals under study. Key words: ringworm (trichophytosis), inactivated vaccine, prophylaxis, treatment, delayed
hypersensitivity test, migration inhibition test. Rrsum~-L'etficacit6 d'un nouveau vaccin inactiv6contre la trichophytie du brtail a 6t6 6tudire sur des cobayes et des veaux dans des conditions exprrimentales et sur 3 troupeaux de b&ail dans des conditions de contagion naturelle. Le vaccin contenant la souche Trichophyton verrucosum a entrain6 une nette rrponse immunologique du type cellulaire. La rraction immunologique, apprrcire ~i l'aide du test d'inhibition de migration du leucocytes etait en correlation avec l'immunitr,drtermin~e par epreuve infestante. Dans les troupeaux off 30 ~i670 d'animaux ont ~t~ infestrs avec la souche T. verrucosum on a constat6 aprrs 4 semaines, aprrs avoir requ deux doses de vaccin, une diminution du nombre de'animaux prrsentant des sympt6mes cliniques de maladie dans la proposition de 40,5% a 100% par rapport au temoins. Mots-clefs: trichophytie/trichophytosis, vaccin inactivr, prophylaxie, traitement, test d'hypersensibilit6 tardive, test d'inhibition de migration.
INTRODUCTION R i n g w o r m (trichophytosis) is a c o m m o n debilitating disease of animals, chiefly of cattle, which is economically i m p o r t a n t to the breeder because it entails loss o f p r o d u c t i o n , costs o f treatment, a n d a b a n o n export. A n i m a l s trichophytosis is also a n i m p o r t a n t p r o b l e m for public health due to its easy t r a n s m i s s i o n to m a n [l~t]. M o d e r n c o n t r o l of the disease in a n i m a l s consists in a d m i n i s t e r i n g different i m m u n o p r e p a r a t i o n s . U p till n o w some vaccines have been developed a n d / o r applied in veterinary practice against r i n g w o r m in cattle [5-10], in horses [11], a n d in fur a n i m a l s [12, 13]. All these p r e p a r a t i o n s are m o n o v a l e n t alive vaccines which, however, have short storage life if n o t lyophilized. I n addition, all these strains in the vaccines, like other a t t e n u a t e d strains, are d a n g e r o u s for individuals with i m m u n o l o g i c a l defects; they can also revert to virulent forms a n d therefore m a y constitute some d a n g e r o f infection for h u m a n beings. In order to avoid all these disadvantages a new sort o f vaccine has been developed by the selection o f a highly i m m u n o g e n i c strain o f T r i c h o p h y t o n v e r r u c o s u m a n d its i n a c t i v a t i o n u n d e r proper conditions. Tests o n the vaccine activity have been carried out o n guinea pigs a n d calves u n d e r strictly controlled c o n d i t i o n s a n d o n cattle u n d e r field c o n d i t i o n s to assess n o t only the prophylactic b u t also the therapeutic value o f the vaccine. 31
32
K. WAWRZKIEWICZand J. WAWRZKIEWICZ MATERIALS AND METHODS
Vaccine. A suspension of T. verrucosum (Tv) strain No. 43, selected on the basis of its antigenic structure [14], was used to prepare the vaccine according to our own method [15]. Briefly, the strain producing spores abundantly was grown in Sabouraud's liquid medium and its culture suspension containing approx. 5 x l 0 7 C F U ml ~ was inactivated with formalin (0.3%). After checking its sterility and harmlessness on mice and guinea pigs the vaccine was administered intramuscularly. Immunisation Guinea pigs. Each of 18 guinea pigs, free from mycotic dermatoses, was injected once with 0.5 ml of the vaccine. An immune response was evaluated by the allergic skin (AST) and the migration inhibition test (MIT) in 12 animals. The other 6 vaccinated individuals were exposed to the "challenge" test of virulent strains of T. verrucosum and T. mentagrophytes. Each animal was administered the virulent strains (105 C F U ml 1) by rubbing into both sides of the body. Six unvaccinated guinea pigs served as control animals. Calves aged 5 - 8 days. The first group of animals (I) comprised 4 experimental calves and 1 control; they were vaccinated in autumn at a low air temperature and low relative humidity (58 67%) in the stalls. The second group, containing 6 experimental calves and 2 control animals (II), was vaccinated in spring at a fairly high air temperature (20-22°C) and a high relative humidity (83-91%) of the surroundings. In the first days of their life some of the animals were given colostrum in the amount of 3 6 litres daily and others from 4.5 to 9 1. Calves aged 1-3 months. Studies were also performed in two seasons altogether on 15 calves of the lowland black and white breed. The first experiment was carried out on 4 calves and 2 controls in the autumn-winter period, and the second on 6 vaccinated and 3 control animals in the spring-summer period. The animals were being housed for 4 months in separate stalls, each for 2--3 animals. The vaccine was given twice, at intervals of 14 days at a dose from 5 to 8 ml depending on the animals age. Evaluation of immunity under field conditions Studies were performed on cattle in the autumn winter period under natural conditions of infection. The animals were housed together in an infected environment where trichophytosis had been recorded. The observations concerned 639 vaccinated animals of different age, some of which showed the ringworm signs confirmed by mycological examinations. Seventeen unvaccinated animals with the signs of ringworm served as controls. The lesions were found chiefly on the neck and head, but a general dissemination over the body occurred as well. For prophylactic purposes the vaccine was administered according to Aamodt et al. [16] at the dose from 5 to 8 ml, twice, 14 days apart, and in animals with mycotic lesions the doses were 50% higher. The efficacy of the preparation was assayed on the basis of lesions recession and the percentage of cured animals.
Allergic skin test (AST) A filtrate of a 4 months-old culture of the T. mentagrophytes 1 strain in Sabouraud's liquid medium with 4% of dextrose, containing 3.4 g ml ~ protein and 34.5 g ml t sugar, was used as the allergen. Guinea pigs were injected with 0.1 ml of the allergen intracutaneously in the scapula region. The results were read after 24 and 48 h taking into consideration the diameter of redness, swelling and necrosis. In the case of the cattle, the allergen was administered in the neck region at a dose of 0.4 ml and the results were
An inactivated vaccine against ringworm
33
assessed after 24h. An increase of at least 3 mm of the skin fold thickness over the prechallenge state was regarded as a positive result. Migration inhibition test ( M I T )
The test was performed in capillaries according to the method described earlier by Wawrzkiewicz and Zi6tkowska [17]. The source of sensitized cells in guinea pigs was the spleen and peripheral blood in the case of the cattle. A suspension of leukocytes was prepared after the method of Moreno-Lopez [18]. Trichophytin as a specific antigen was prepared from the T. mentagrophytes 1 strain and applied at a dose of 0.005 ml ~ of the medium. Average values of migration areas of cells were calculated on the basis of 15 cultures with the antigen and 15 cultures without the antigen. Migration inhibition was calculated from the following equation: 1 -
average migration area with antigen average migration area without antigen
x
100.
Taking into account the findings from in control animals a significant degree of cell migration inhibition was accepted to be 20% or more. Challenge test
An experimental infection of guinea pigs was done 6 weeks after vaccination for 2 consecutive days by rubbing a dense suspension of virulent strains into the depilated and scarified skin in the dorsal region. T. mentagrophytes was administered on one side and T. verrucosum on the other side of each animal. The injected guinea pigs were observed for 4 weeks and the material with pathological changes was assayed mycologically. Calves were experimentally infected 8 weeks after receiving the first dose of the vaccine. A standardized suspension of the T. verrucosum Z strain, containing from 105 to 10 3 CFU ml i, was rubbed for 3 successive days into the skin depilated and scarified on both sides of each animal. The animals were observed for 2 months and all cases of clinical lesions were confirmed by mycological examinations. Statistics
The values of the allergic skin test and migration inhibition test (Table 2) were characterized by the mean (M) and standard deviation (SD). The significance of differences between the means was checked by the Student's t-test or Cochran and Cox's test [19]. The effects of prophylactic and therapeutic action of the inactivated vaccine in infected herds with ringworm were expressed in percentages; the significance of differences between the percentages was checked by Z 2 test (Table 4). A 5% risk of conclusion (P < 0.05) was accepted. RESULTS Immune response in guinea pigs
Table 1 shows the immune response of guinea pigs estimated by the allergic skin test (AST), the migration inhibition test (MIT), and the results of challenge with virulent T. verrucosum and T. mentagrophytes strains. Out of six immunised animals assayed by the allergic skin test, five displayed a delayed type hypersensitivity producing large areas of redness, swelling and/or necrosis at the site of the allergen injection. In one vaccinated guinea pig (No. 10) no changes were noticed, apart from mild redness. In other seven animals immunised and examined by the MIT, with one exception No. 10, the spleen cells migration inhibition was significant, especially on days 14 and 21. Six guinea pigs of the
28
30
R
++
++
7 S
+ +
++
N
25
25
R
+
+
14 S
+
+
N
10~
25
R
+
21 S N
10
R
28 S N
R: redness; S: swelling; N: necrosis; x: pregnant; 1 + to 4 + : intensiveness of clinical lesions.
Controls lc 2c 3c
7 8 9 10 11 12
Guinea pig No.
Allergic test (days)
7
28,4
24.7 62.7 61.6
14
50.0 46.4
21
Migration inhibition test % of inhibition (days)
Table 1. I m m u n e response of guinea pigs vaccinated against ringworm
14.7
28
4c 5c 6c
13 14 15 16 17 18
Guinea pig No.
3+ 2+ 2+
m
E
3+ 3+ 3+
1+ 1+ 1+ I+ 1+
2+
Challenge T.v. T.m. Clinical lesions
N
N
N
e~
N
N
4.5-9.0 4.5-9.0 4.5-9.0 4.5-9.0 4.5-9.0 3.045.0 3.0-6.0 3.0~.0 3.0~.0 3.0~.0 4.5-9.0
3.ff6.0
Calf No.
2 3 4 5 6 7 8 9 10 1-5
6-10
n.t.: not tested.
Controls 11 12 13
1
Daily dose o f collostrum in litres
1.8 1.4
2.0 3.9 2.3 1.7 5.10 +0.86 2.48 ±0.98
6/4+* 11/3+ 3/2 + 17/1 + 132/35/4 + 125/3 + 67/2+ 23/1 + 120/-8/4 + 12/3+ 4/2 + 6/1 + 70/--
37:169
250:370
30:100
I 2-3 months autumn
II 2~, months autumn
II1 7 8 months winter
3/4 + 3/3 + 3/3 + 2/2 +
I1
31/4 + 132/3 + 68/2+ 18/1 + 121/-
1
4/2 + 2/1 + 94/-
14/4+ 3/3+ 2/2 + 18/1 + 132/ 17/4 + 62/3 + 120/2+ 31/1 + 140/-
2
17/4 + 62/3 + 120/2+ 31/I + 140/--
3
4
5/2 +
4/4 + 2/3 + 3/3 + 3/2 +
100/
9/2 + 13/1 + 147/4/4 + 14/3 + 19/2+ 29/1 + 304/-
Clinical status following vaccination Weeks
*Numerator: number of animals; denominator: intensiveness of clinical lesions.
III
6/4 +
1
Controls
Clinical status before vaccination
100
73.6
40.5
% of cured animals after 4 weeks
Table 4. Effects of inactivated vaccine on the course of ringworm in cattle--prophylaxis and treatment
Number of infected animals in relation to the whole herd
Group age of animals and season of observation
10.4
16.6
X2
