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Cutaneous responses of cattle to extracts from Rhipicephalus appendiculatus larvae
Veterinary Parasitology, 15 (1984) 67--73 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
67
C U T A N E O U S R E S P O N S E S OF CATTLE TO EXTRACTS FROM R H I P I C E P H A L US A P P E N D I C U L A T U S L A R V A E
M.G. BINTA and M.P. CUNNINGHAM
International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobz (Kenya) (Accepted for publication 18 October 1983)
ABSTRACT Binta, M.G. and Cunningham, M.P., 1984. Cutaneous responses of cattle to extracts from Rhipzcephalus append~culatus larvae. Vet. Parasztol , 15' 67--73. A component of an extract from Rh~picephalus appendiculatus larval ticks induced an immediate hypersensitivity reaction in sensitized cattle when intra-dermally inoculated. Generally, the magnitude of the cutaneous reaction depended upon the duration of exposure of the cattle to tick infestation. The skin reaction was absent in steers not previously fed on by ticks. Field trials, using the extract in the Narok District of Kenya (Masailand), showed that the skin swelling thus induced was biggest in adult cattle and those over 9 months, moderate in the 6-month age-group and barely palpable in the less-than-l-month age-group.
INTRODUCTION C u t a n e o u s responses o f animals t o tick i n f e s t a t i o n lead to t h e e x e r t i o n o f d e l e t e r i o u s effects o n t h e tick with s u b s e q u e n t rejection (Wikel, 1979). It has been suggested t h a t the process is an i m m u n o l o g i c a l p h e n o m e n o n (Wikel, et al., 1 9 7 8 ) . In cattle, this p h e n o m e n o n has been d e m o n s t r a t e d against B o o p h i l u s m i c r o p l u s ( R o b e r t s 1 9 6 8 a , b) and in guinea pigs against D e r m a c e n t o r species (Allen, 1973). Larval a n d egg extracts f r o m B. m i c r o p l u s and o t h e r i x o d i d ticks have been s h o w n t o i n d u c e an i m m e d i a t e h y p e r s e n s i t i v i t y r e a c t i o n in l a b o r a t o r y animals p r e v i o u s l y e x p o s e d t o these ticks (Riek, 1 9 5 4 ) . H o w e v e r , Riek (1954} did n o t relate the skin r e a c t i o n t o tick exp o s u r e , whereas Willadsen et al. ( 1 9 7 8 ) were able t o d o this. It has also been observed in o u r l a b o r a t o r y t h a t w h e n cattle are e x p o s e d to the tick Rhipicephalus a p p e n d i c u l a t u s , t h e y acquire v a r y i n g degrees o f resistance t o its instars. T h e aims o f o u r investigation were t o p r o d u c e an e x t r a c t f r o m R. appendzculatus w h i c h c o u l d be used in an intra-dermal test to i d e n t i f y cattle p r e v i o u s l y e x p o s e d t o R. a p p e n d i c u l a t u s .
0304-4017/84/$03.00
O 1984 Elsevier Science Publishers B.V.
68 MATERIALS AND METHODS Animals Four y o u n g Fnesian steers and 11 grade steers were tested. The Friesian steers had no previous tick exposure as they had been reared from birth under tick-free conditions. The rest of the animals had been exposed to ticks naturally in the field. For the field trials, 15 African short-horn zebu (Masai} cattle were used in 3-month, 6-month and less-than-l-month agegroups each consisting of 5 animals. Source o f antzgen An antigenic extract was prepared from 6.5 g of unfed R. appendwulatus larvae 14 days after hatching. Briefly, larvae in 2 ml PBS per gram of ticks were ground in a mortar placed on an ice bath. The homogenate was sonicated in an ultrasonic transducer (Dawe Instruments, U.K.) at a frequency of 41.23 kc s -~ for 3 s. The larval homogenate was then separated by centrifugation 4 times at 3000 r.p.m, for 30 min, after which time the supernatant was retained. The supernatant was precipitated with 33% (w/v) a m m o n i u m sulphate at --4°C overnight with constant stirring. The amm o n i u m sulphate--larval homogenate mixture was centrifuged at 3000 r.p.m. for 15 min. The precipitate thus obtained was reconstituted with 10 ml phosphate buffered saline (PBS) and then dialyzed against 3 changes of Tris-HC1 buffer (0.05 M pH 8.7) overnight at 4°C. The product of dialysis was then loaded on to a column of DEAE--cellulose (DE52) (Pharmacia Chemicals, Sweden) equilibrated with Tris-HC1 buffer 0.05 M, pH 8.7. The components were eluted stepwise using Tris-HC1 buffer (0.05 M, pH 8.7) containing 0.05 M, 0.1 M and 0.2 M sodium chloride. The eluted protem peaks were collected separately and concentrated with polyethylene glycol 6000 (BDH) for 3 h. This material was reconstituted in sodium acetate buffer 0.05 M, pH 4.6). The larval extract protein peaks were applied separately to a column of CM-cellulose equilibrated in sodmm acetate buffer (0.05 M, pH 4.6) containing 0.05 M, 0.1 M and 0.2 M sodium chloride. The material thus obtained was finally loaded on a column (2.5 × 25 cm) of sephadex G-100 superfine (Pharmacia Chemicals, Sweden) and then eluted with Tris-HC1 buffer (0.1 M Tris-HC1, pH 7.5). The protein concentration of the peaks was determined according to the m e t h o d of Lowry et al. (1951}. In tra-dermal test An area in the middle of the neck (10 cm ~ ) on either side was shaved and cleaned with ethanol. The mean skin thickness in 2 dimensions was determined at different points using a pair of tuberculin callipers. Three sites were chosen within the shaved area; one was intra-cutaneously injected with
69 0.1 ml of a 10 -1 dilution of extract Peak 2 (0.86 mg ml -~ protein), and the control site was injected with PBS (pH 7.2). The changes in the skin thickness were observed and reported after 30 min as recommended by Riek (1954). The mean diameter of the resulting lesions was measured using a ruler (Table I). Skin biopsies were taken from the centre of the skin reaction, fixed in 10% formalin and then stained with h a e m o t o x y l i n and eosin. The dose--response curve of Peak 2 or Allergen 2 was obtained by titrating the extract in a highly tick resistant steer (Fig. 1).
0 300
< 200 z
IOC
-3 10
-2 I0
-I P(D
DILLITIO['4 A L L E R ~ S E N
0 I0 2
Fig. 1. D o s e - - r e s p o n s e curve o f tick larval-extract (Allergen 2) t i t r a t e d m the skin o f a highly tick resistant steer. N o t e : a and b = radii o f the elliptical skin swelling in 2 d i m e n sions.
F~eld trials Field trials were conducted in the Narok District of Kenya (Masailand) using 15 African short-horn zebu cattle. Calves of either sex were allocated to 9-month, 6-month and less-than-l-month age-groups, each consisting of 5 animals. The calves less than 1 m o n t h of age were kept in the cattle bomas (cattle shed) within the m a n y a t t a (traditional Masai homestead). Rhipzcephalus appendiculatus mstars were shown to be present on the pastures grazed by the 9-month and 6-month age-groups. The skin test was performed as described above. Attempts were made to relate the size of the skin swelling and the animal age group.
70
Laboratory paddock trials F o u r exotic breed-type steers with minimal previous tick exposure were p u t o n t o pastures seeded with the instars o f R. append~culatus. However, the p o p u l a t i o n of the ticks on the pastures was n o t assessed. The intradermal skin test was p e r f o r m e d on the animals before i n t r o d u c t i o n to the p a d d o c k pastures and f r om then on at weekly intervals. RESULTS Two peaks o f the extract, referred to as Peak 1 and Peak 2 of protein c o n c e n t r a t i o n 1.08 mg m1-1 and 0.86 mg ml -I , respectively, were obtained f r o m the crude larval e x t r a c t as shown in Figs 2--4 using ion-exchange chrom a t o g r a p h y . Peak 2, h e n c e f o r t h referred to as All. 2, was able to cause o e d e m a t o u s swellings in the skin of tick-exposed, but n o t tick-naive steers. Only the second peak was used in the investigation. The reaction was characterized by a blanching of the injection site within a b o u t 3 s followed by hyperaemia. The hyper a e m i a was m or e obvious in light-skinned animals. Within 5 min, the hyperaemic area became a circumscribed o e d e m a t o u s area. The swelling became wider and indurated with time. During this time, the animal evinced pain on palpation of the swelling. F r e q u e n t urination and swishing o f the tail were c o m m o n di s c omfort signs in the animals elicitmg the skin reaction. A papular rash sometimes a c c o m p a m e d the reaction and
OOSM
IMNac{IMNacl
~
10
o08.
PEAK 1
~0.6, m ~04. 2
02.
2
3
2.
FRACTION NUMBER
~,
i5
~,_
Fig. 2. Elution profile of crude Rhzpicephalus appendzculatus extract on DE-52 Tris-HCl buffer, 0.05 M, pH 8.7, with stepwise gradient of sodium chloride.
71
was n o t co n f in ed to the vicinity o f the swelling. The o e d e m a t o u s skin reaction which was maximal at 30 min started waning t h e r e a f t e r and by 6-h had externally disappeared. It was n o t e d t h a t this reaction was absent in tick-naive calves and at the PBS injection sites. Table I shows the skin changes, i.e., mean diameter and thickness of the lesion.
10. E c
o
~,U z
06-
~
04.
Ixl <
L_.__
0~-
;,
~ ; ,'o FRACTION NUMBER
1'2
'4
Fig. 3. Elution profile of Peak 1 on CM-cellulose using acetate buffer, 0.05 M, pH 4.5.
t o
I.o
I-
< o,8 U,l U
~ 06 czl nr
o
m (14 <
0.2
1 2 3 4 5
6
7
8
g
10 11 1 2 1 3
FRACTION N U M B E R
1/. 15 16 1 7 1 8
lg 2 0 2 1
22
~-
Fig. 4. Elution profile,of Peak 2 on CM-cellulose using acetate buffer, 0.05 M, pH 4.5.
72 TABLE I Diameter of swelling and increase in skin thickness after intra-dermal inoculation of the extract, Peak 2.
Grade steers Boran steers Friesian calves
Mean dlamater of reaction in skin ± s.d. (ram)
Mean increase in skin thickness -+ s.d. (mm)
33.0 *- 5.2 (n=11) 15.0 ± 2.0 (n=5) --* (n=4)
14.0 * 1.5 4.0 ± 1.2 --*
*No swelling observed and therefore no increase in skin thmkness. H i s t o l o g m a l skin s e c t i o n s o f t h e e x t r a c t i n j e c t i o n sites, t a k e n 30 m i n a n d 6 h p o s t e x t r a c t - i n j e c t i o n a n d s t a i n e d w i t h h a e m o t o x y l i n a n d eosin, s h o w e d a p r e p o n d e r a n c e o f p o l y m o r p h o n u c l e a r l e u c o c y t e s , m a i n l y n e u t r o p h i l s , in the dermis. A f e w e o s i n o p h i l s w e r e p r e s e n t 30 m i n p o s t e x t r a c t - i n o c u l a t i o n , b u t t h e i r n u m b e r s h a d increased at 6 h p o s t e x t r a c t - i n j e c t i o n . O e d e m a o f t h e e p i d e r m i s a n d d e r m i s w e r e also p r o m i n e n t at 30 m m , b u t r e c e d e d w i t h t i m e . H i s t o l o g y o f skin e x t r a c t - i n j e c t i o n sites in t i c k - n a i v e a n i m a l s s h o w e d o n l y a f e w p o l y m o r p h o n u c l e a r l e u c o c y t e s m t h e dermis. No o t h e r changes w e r e evident. T h e s e l e u c o c y t e s w e r e also o b s e r v e d in t h e skin swelling r e d u c e d b y larval t i c k - e x t r a c t i n o c u l a t i o n m a n i m a l s t h a t h a d been subject to more tick exposure than those moderately exposed. W h e n t h e e x t r a c t was i n j e c t e d into calves o f d i f f e r e n t age groups, 1.e., aged 9 m o n t h s , 6 m o n t h s a n d less t h a n 1 m o n t h , t h e size o f the skin r e a c t i o n was d e p e n d e n t u p o n t h e a m m a l ' s age. In o t h e r w o r d s , t h e skin swelling m t h e calves less t h a n 1 m o n t h old was e i t h e r a b s e n t or b a r e l y p a l p a b l e . T h e average d i a m e t e r s o f t h e s e swellings in t h e 9 - m o n t h - a n d 6 - m o n t h - o l d calves w e r e 40 a n d 30 m m , respectively. In t h e l a b o r a t o r y p a d d o c k trials, d e v e l o p m e n t o f sensitivity t o t h e trek allergens was variable. DISCUSSION P e a k 2 o b t a i n e d b y 1on-exchange c h r o m a t o g r a p h y was s h o w n t o i n d u c e a swelling u p o n i n t r a - d e r m a l i n o c u l a t i o n i n t o t i c k - e x p o s e d cattle, w h i c h c o n f o r m e d to a T y p e 1 h y p e r s e n s i t i v i t y r e a c t i o n . T h e initial e r y t h e m a t o u s a n d o e d e m a t o u s r e s p o n s e was suggestive o f a local i m m e d i a t e h y p e r s e n s i t i v ity r e a c t i o n d e s i g n a t e d as T y p e 1 b y Gell et al. (1975). T h e d i l u t i o n o f 10 -~ was a d o p t e d a f t e r t i t r a t i o n in a t i c k - r e s i s t a n t a n d tick-naive a n i m a l t o o b t a i n a d o s e - - r e s p o n s e curve. T h e field trial results suggest t h a t t h e skin t e s t c o u l d f o r m a p r o m i s i n g diagnostic t o o l f o r i d e n t i f y i n g c a t t l e p r e v i o u s l y i n f e s t e d a n d h e n c e sensitized b y ticks. T h e t e s t is easy t o a p p l y u n d e r field a n d l a b o r a t o r y c o n d i t i o n s , q u i c k t o r e a d a n d t i t r a t i o n o f t h e e x t r a c t is n o t r e q u i r e d as m a n y doses can
73
be produced from one batch. Also, no loss of allergenic p o t e n c y has been observed at --20°C after 24 months. It is hoped that the test could be quantitated to measure the tick resistance status of cattle. ACKNOWLEDGEMENTS
This paper was published with the permission of the Director of the International Centre for Insect Physiology and Ecology, Nairobi, Kenya. We are grateful to the Director of Veterinary Research Department, Muguga, for providing the facilities to do this work.
REFERENCES Allen, J.R., 1973. Tick resistance: basophils in skin reactions of resistant guinea pigs. Int. J. Parasitol., 3: 195--200. Gell, P.H.G., Coombs, R.R.A. and Lachmann, R. (Editors), 1975. Clinical Aspects of Immunology. 3rd edn., Section IV, Blackwell Scientific, Oxford. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin reagent. J. Biol. Chem., 193: 256--275. Riek, R.F., 1954. Studies of ixodid ticks, a preliminary note on the reaction in laboratory animals following parenteral administration of egg and larval extracts. Trans. R. Soc. Trop. Med. Hyg., 48: 286. Roberts, J.A., 1968a. Acquisition by the host of resistance to the cattle tick, Boophllus mlcroplus (Canestrini). J. Parasitol., 54: 657--662. Roberts, J.A., 1968b. Resistance of cattle to the tick Boophilus microplus (Canestrlni). I. Development of ticks on Bos taurus. J. Parasitol., 54: 663--666. Wikel, S.K., 1979. Acquired resistance to ticks. Expression of resistance by C4 deficient guinea pigs. Am. J. Trop. Med. Hyg., 28: 586--590. Wikel, S.K., Graham, J.E. and Allen, J.R., 1978. Acquired resistance to ticks. IV. Skin reactivity and in vitro lymphocyte responsiveness to salivary gland antigen. Am. J. Trop. Med. Hyg., 34: 257--263. Willadsen, P., Williams, P.G., Roberts, J.A. and Kerr, J.D., 1978. Response of cattle to allergens from Boophilus microplus Int. J. Parasitol., 8: 89--95.
67
C U T A N E O U S R E S P O N S E S OF CATTLE TO EXTRACTS FROM R H I P I C E P H A L US A P P E N D I C U L A T U S L A R V A E
M.G. BINTA and M.P. CUNNINGHAM
International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobz (Kenya) (Accepted for publication 18 October 1983)
ABSTRACT Binta, M.G. and Cunningham, M.P., 1984. Cutaneous responses of cattle to extracts from Rhipzcephalus append~culatus larvae. Vet. Parasztol , 15' 67--73. A component of an extract from Rh~picephalus appendiculatus larval ticks induced an immediate hypersensitivity reaction in sensitized cattle when intra-dermally inoculated. Generally, the magnitude of the cutaneous reaction depended upon the duration of exposure of the cattle to tick infestation. The skin reaction was absent in steers not previously fed on by ticks. Field trials, using the extract in the Narok District of Kenya (Masailand), showed that the skin swelling thus induced was biggest in adult cattle and those over 9 months, moderate in the 6-month age-group and barely palpable in the less-than-l-month age-group.
INTRODUCTION C u t a n e o u s responses o f animals t o tick i n f e s t a t i o n lead to t h e e x e r t i o n o f d e l e t e r i o u s effects o n t h e tick with s u b s e q u e n t rejection (Wikel, 1979). It has been suggested t h a t the process is an i m m u n o l o g i c a l p h e n o m e n o n (Wikel, et al., 1 9 7 8 ) . In cattle, this p h e n o m e n o n has been d e m o n s t r a t e d against B o o p h i l u s m i c r o p l u s ( R o b e r t s 1 9 6 8 a , b) and in guinea pigs against D e r m a c e n t o r species (Allen, 1973). Larval a n d egg extracts f r o m B. m i c r o p l u s and o t h e r i x o d i d ticks have been s h o w n t o i n d u c e an i m m e d i a t e h y p e r s e n s i t i v i t y r e a c t i o n in l a b o r a t o r y animals p r e v i o u s l y e x p o s e d t o these ticks (Riek, 1 9 5 4 ) . H o w e v e r , Riek (1954} did n o t relate the skin r e a c t i o n t o tick exp o s u r e , whereas Willadsen et al. ( 1 9 7 8 ) were able t o d o this. It has also been observed in o u r l a b o r a t o r y t h a t w h e n cattle are e x p o s e d to the tick Rhipicephalus a p p e n d i c u l a t u s , t h e y acquire v a r y i n g degrees o f resistance t o its instars. T h e aims o f o u r investigation were t o p r o d u c e an e x t r a c t f r o m R. appendzculatus w h i c h c o u l d be used in an intra-dermal test to i d e n t i f y cattle p r e v i o u s l y e x p o s e d t o R. a p p e n d i c u l a t u s .
0304-4017/84/$03.00
O 1984 Elsevier Science Publishers B.V.
68 MATERIALS AND METHODS Animals Four y o u n g Fnesian steers and 11 grade steers were tested. The Friesian steers had no previous tick exposure as they had been reared from birth under tick-free conditions. The rest of the animals had been exposed to ticks naturally in the field. For the field trials, 15 African short-horn zebu (Masai} cattle were used in 3-month, 6-month and less-than-l-month agegroups each consisting of 5 animals. Source o f antzgen An antigenic extract was prepared from 6.5 g of unfed R. appendwulatus larvae 14 days after hatching. Briefly, larvae in 2 ml PBS per gram of ticks were ground in a mortar placed on an ice bath. The homogenate was sonicated in an ultrasonic transducer (Dawe Instruments, U.K.) at a frequency of 41.23 kc s -~ for 3 s. The larval homogenate was then separated by centrifugation 4 times at 3000 r.p.m, for 30 min, after which time the supernatant was retained. The supernatant was precipitated with 33% (w/v) a m m o n i u m sulphate at --4°C overnight with constant stirring. The amm o n i u m sulphate--larval homogenate mixture was centrifuged at 3000 r.p.m. for 15 min. The precipitate thus obtained was reconstituted with 10 ml phosphate buffered saline (PBS) and then dialyzed against 3 changes of Tris-HC1 buffer (0.05 M pH 8.7) overnight at 4°C. The product of dialysis was then loaded on to a column of DEAE--cellulose (DE52) (Pharmacia Chemicals, Sweden) equilibrated with Tris-HC1 buffer 0.05 M, pH 8.7. The components were eluted stepwise using Tris-HC1 buffer (0.05 M, pH 8.7) containing 0.05 M, 0.1 M and 0.2 M sodium chloride. The eluted protem peaks were collected separately and concentrated with polyethylene glycol 6000 (BDH) for 3 h. This material was reconstituted in sodium acetate buffer 0.05 M, pH 4.6). The larval extract protein peaks were applied separately to a column of CM-cellulose equilibrated in sodmm acetate buffer (0.05 M, pH 4.6) containing 0.05 M, 0.1 M and 0.2 M sodium chloride. The material thus obtained was finally loaded on a column (2.5 × 25 cm) of sephadex G-100 superfine (Pharmacia Chemicals, Sweden) and then eluted with Tris-HC1 buffer (0.1 M Tris-HC1, pH 7.5). The protein concentration of the peaks was determined according to the m e t h o d of Lowry et al. (1951}. In tra-dermal test An area in the middle of the neck (10 cm ~ ) on either side was shaved and cleaned with ethanol. The mean skin thickness in 2 dimensions was determined at different points using a pair of tuberculin callipers. Three sites were chosen within the shaved area; one was intra-cutaneously injected with
69 0.1 ml of a 10 -1 dilution of extract Peak 2 (0.86 mg ml -~ protein), and the control site was injected with PBS (pH 7.2). The changes in the skin thickness were observed and reported after 30 min as recommended by Riek (1954). The mean diameter of the resulting lesions was measured using a ruler (Table I). Skin biopsies were taken from the centre of the skin reaction, fixed in 10% formalin and then stained with h a e m o t o x y l i n and eosin. The dose--response curve of Peak 2 or Allergen 2 was obtained by titrating the extract in a highly tick resistant steer (Fig. 1).
0 300
< 200 z
IOC
-3 10
-2 I0
-I P(D
DILLITIO['4 A L L E R ~ S E N
0 I0 2
Fig. 1. D o s e - - r e s p o n s e curve o f tick larval-extract (Allergen 2) t i t r a t e d m the skin o f a highly tick resistant steer. N o t e : a and b = radii o f the elliptical skin swelling in 2 d i m e n sions.
F~eld trials Field trials were conducted in the Narok District of Kenya (Masailand) using 15 African short-horn zebu cattle. Calves of either sex were allocated to 9-month, 6-month and less-than-l-month age-groups, each consisting of 5 animals. The calves less than 1 m o n t h of age were kept in the cattle bomas (cattle shed) within the m a n y a t t a (traditional Masai homestead). Rhipzcephalus appendiculatus mstars were shown to be present on the pastures grazed by the 9-month and 6-month age-groups. The skin test was performed as described above. Attempts were made to relate the size of the skin swelling and the animal age group.
70
Laboratory paddock trials F o u r exotic breed-type steers with minimal previous tick exposure were p u t o n t o pastures seeded with the instars o f R. append~culatus. However, the p o p u l a t i o n of the ticks on the pastures was n o t assessed. The intradermal skin test was p e r f o r m e d on the animals before i n t r o d u c t i o n to the p a d d o c k pastures and f r om then on at weekly intervals. RESULTS Two peaks o f the extract, referred to as Peak 1 and Peak 2 of protein c o n c e n t r a t i o n 1.08 mg m1-1 and 0.86 mg ml -I , respectively, were obtained f r o m the crude larval e x t r a c t as shown in Figs 2--4 using ion-exchange chrom a t o g r a p h y . Peak 2, h e n c e f o r t h referred to as All. 2, was able to cause o e d e m a t o u s swellings in the skin of tick-exposed, but n o t tick-naive steers. Only the second peak was used in the investigation. The reaction was characterized by a blanching of the injection site within a b o u t 3 s followed by hyperaemia. The hyper a e m i a was m or e obvious in light-skinned animals. Within 5 min, the hyperaemic area became a circumscribed o e d e m a t o u s area. The swelling became wider and indurated with time. During this time, the animal evinced pain on palpation of the swelling. F r e q u e n t urination and swishing o f the tail were c o m m o n di s c omfort signs in the animals elicitmg the skin reaction. A papular rash sometimes a c c o m p a m e d the reaction and
OOSM
IMNac{IMNacl
~
10
o08.
PEAK 1
~0.6, m ~04. 2
02.
2
3
2.
FRACTION NUMBER
~,
i5
~,_
Fig. 2. Elution profile of crude Rhzpicephalus appendzculatus extract on DE-52 Tris-HCl buffer, 0.05 M, pH 8.7, with stepwise gradient of sodium chloride.
71
was n o t co n f in ed to the vicinity o f the swelling. The o e d e m a t o u s skin reaction which was maximal at 30 min started waning t h e r e a f t e r and by 6-h had externally disappeared. It was n o t e d t h a t this reaction was absent in tick-naive calves and at the PBS injection sites. Table I shows the skin changes, i.e., mean diameter and thickness of the lesion.
10. E c
o
~,U z
06-
~
04.
Ixl <
L_.__
0~-
;,
~ ; ,'o FRACTION NUMBER
1'2
'4
Fig. 3. Elution profile of Peak 1 on CM-cellulose using acetate buffer, 0.05 M, pH 4.5.
t o
I.o
I-
< o,8 U,l U
~ 06 czl nr
o
m (14 <
0.2
1 2 3 4 5
6
7
8
g
10 11 1 2 1 3
FRACTION N U M B E R
1/. 15 16 1 7 1 8
lg 2 0 2 1
22
~-
Fig. 4. Elution profile,of Peak 2 on CM-cellulose using acetate buffer, 0.05 M, pH 4.5.
72 TABLE I Diameter of swelling and increase in skin thickness after intra-dermal inoculation of the extract, Peak 2.
Grade steers Boran steers Friesian calves
Mean dlamater of reaction in skin ± s.d. (ram)
Mean increase in skin thickness -+ s.d. (mm)
33.0 *- 5.2 (n=11) 15.0 ± 2.0 (n=5) --* (n=4)
14.0 * 1.5 4.0 ± 1.2 --*
*No swelling observed and therefore no increase in skin thmkness. H i s t o l o g m a l skin s e c t i o n s o f t h e e x t r a c t i n j e c t i o n sites, t a k e n 30 m i n a n d 6 h p o s t e x t r a c t - i n j e c t i o n a n d s t a i n e d w i t h h a e m o t o x y l i n a n d eosin, s h o w e d a p r e p o n d e r a n c e o f p o l y m o r p h o n u c l e a r l e u c o c y t e s , m a i n l y n e u t r o p h i l s , in the dermis. A f e w e o s i n o p h i l s w e r e p r e s e n t 30 m i n p o s t e x t r a c t - i n o c u l a t i o n , b u t t h e i r n u m b e r s h a d increased at 6 h p o s t e x t r a c t - i n j e c t i o n . O e d e m a o f t h e e p i d e r m i s a n d d e r m i s w e r e also p r o m i n e n t at 30 m m , b u t r e c e d e d w i t h t i m e . H i s t o l o g y o f skin e x t r a c t - i n j e c t i o n sites in t i c k - n a i v e a n i m a l s s h o w e d o n l y a f e w p o l y m o r p h o n u c l e a r l e u c o c y t e s m t h e dermis. No o t h e r changes w e r e evident. T h e s e l e u c o c y t e s w e r e also o b s e r v e d in t h e skin swelling r e d u c e d b y larval t i c k - e x t r a c t i n o c u l a t i o n m a n i m a l s t h a t h a d been subject to more tick exposure than those moderately exposed. W h e n t h e e x t r a c t was i n j e c t e d into calves o f d i f f e r e n t age groups, 1.e., aged 9 m o n t h s , 6 m o n t h s a n d less t h a n 1 m o n t h , t h e size o f the skin r e a c t i o n was d e p e n d e n t u p o n t h e a m m a l ' s age. In o t h e r w o r d s , t h e skin swelling m t h e calves less t h a n 1 m o n t h old was e i t h e r a b s e n t or b a r e l y p a l p a b l e . T h e average d i a m e t e r s o f t h e s e swellings in t h e 9 - m o n t h - a n d 6 - m o n t h - o l d calves w e r e 40 a n d 30 m m , respectively. In t h e l a b o r a t o r y p a d d o c k trials, d e v e l o p m e n t o f sensitivity t o t h e trek allergens was variable. DISCUSSION P e a k 2 o b t a i n e d b y 1on-exchange c h r o m a t o g r a p h y was s h o w n t o i n d u c e a swelling u p o n i n t r a - d e r m a l i n o c u l a t i o n i n t o t i c k - e x p o s e d cattle, w h i c h c o n f o r m e d to a T y p e 1 h y p e r s e n s i t i v i t y r e a c t i o n . T h e initial e r y t h e m a t o u s a n d o e d e m a t o u s r e s p o n s e was suggestive o f a local i m m e d i a t e h y p e r s e n s i t i v ity r e a c t i o n d e s i g n a t e d as T y p e 1 b y Gell et al. (1975). T h e d i l u t i o n o f 10 -~ was a d o p t e d a f t e r t i t r a t i o n in a t i c k - r e s i s t a n t a n d tick-naive a n i m a l t o o b t a i n a d o s e - - r e s p o n s e curve. T h e field trial results suggest t h a t t h e skin t e s t c o u l d f o r m a p r o m i s i n g diagnostic t o o l f o r i d e n t i f y i n g c a t t l e p r e v i o u s l y i n f e s t e d a n d h e n c e sensitized b y ticks. T h e t e s t is easy t o a p p l y u n d e r field a n d l a b o r a t o r y c o n d i t i o n s , q u i c k t o r e a d a n d t i t r a t i o n o f t h e e x t r a c t is n o t r e q u i r e d as m a n y doses can
73
be produced from one batch. Also, no loss of allergenic p o t e n c y has been observed at --20°C after 24 months. It is hoped that the test could be quantitated to measure the tick resistance status of cattle. ACKNOWLEDGEMENTS
This paper was published with the permission of the Director of the International Centre for Insect Physiology and Ecology, Nairobi, Kenya. We are grateful to the Director of Veterinary Research Department, Muguga, for providing the facilities to do this work.
REFERENCES Allen, J.R., 1973. Tick resistance: basophils in skin reactions of resistant guinea pigs. Int. J. Parasitol., 3: 195--200. Gell, P.H.G., Coombs, R.R.A. and Lachmann, R. (Editors), 1975. Clinical Aspects of Immunology. 3rd edn., Section IV, Blackwell Scientific, Oxford. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin reagent. J. Biol. Chem., 193: 256--275. Riek, R.F., 1954. Studies of ixodid ticks, a preliminary note on the reaction in laboratory animals following parenteral administration of egg and larval extracts. Trans. R. Soc. Trop. Med. Hyg., 48: 286. Roberts, J.A., 1968a. Acquisition by the host of resistance to the cattle tick, Boophllus mlcroplus (Canestrini). J. Parasitol., 54: 657--662. Roberts, J.A., 1968b. Resistance of cattle to the tick Boophilus microplus (Canestrlni). I. Development of ticks on Bos taurus. J. Parasitol., 54: 663--666. Wikel, S.K., 1979. Acquired resistance to ticks. Expression of resistance by C4 deficient guinea pigs. Am. J. Trop. Med. Hyg., 28: 586--590. Wikel, S.K., Graham, J.E. and Allen, J.R., 1978. Acquired resistance to ticks. IV. Skin reactivity and in vitro lymphocyte responsiveness to salivary gland antigen. Am. J. Trop. Med. Hyg., 34: 257--263. Willadsen, P., Williams, P.G., Roberts, J.A. and Kerr, J.D., 1978. Response of cattle to allergens from Boophilus microplus Int. J. Parasitol., 8: 89--95.