ELSEVIER Veterinary Parasitology 52 (1994) 79-90

veterinary parasitology

Immunisation of dogs, hamsters and guinea pigs against Rhipicephalus sanguineus using crude unfed

adult tick extracts

G . H . B e c h a r a *'a, M . P . J . S z a b 6 b, L.S. M u k a i a, P .C.S . R o s a ~

aPathology Department, School of Veterinary Science of Jaboticabal, University of the Sdo Paulo State-UNESP, 14870-000, Jaboticabal, SP, Brazil

bpathology Department, School of Veterinary Science, University of SYto Paulo- USP, S~o Paulo, Brazil

(Accepted 20 May 1993 )

Abstract

Naive experimental groups of dogs, hamsters and guinea pigs were inoculated three times subcutaneously with unfed adult extract of the tick Rhipicephalus sanguineus and chal- lenged with adult R. sanguineus to evaluate resistance. The acquisition of resistance was based on alterations of some reproductive and feeding performance parameters of female ticks such as female and egg mass weights, engorgement, pre-oviposition and incubation periods, larval hatchability rate and efficiency rates of female ticks in converting their food reservoir to eggs and larvae. Dogs did not develop resistance under these experimental conditions; guinea pigs and hamsters, to a lesser extent, acquired an effective immunity to ticks as demonstrated by the impairment of the reproductive and feeding performance. However, the resistance induced by inoculation of the extract in the rodents seemed not to be as efficient as that induced by successive infestations.

Key words: Rhipicephalus sanguineus; Dog; Hamster; Guinea pig; Immunization-Arthropoda

1. Introduction

The induct ion o f resistance in hosts against t icks seems to be a p romis ing alter- na t ive in the control o f these ar thropods . Wil ladsen (1987) po in ted out two im- munologica l approaches to achieve this resistance. The first involves the selec-

*Corresponding author: Fax. (0163 ) 224275; Tel. (0163 ) 232500 ext. 117.

0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-4017 (93)00549-E

80 G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90

tion of hosts capable of exhibiting high levels of resistance; the second involves the use of tick antigens to induce resistance by direct immunisation. The latter approach is based on the presentation of tick antigens to the host's immune sys- tem other than those involved in a natural host-parasite contact. This would be a way of avoiding evasion mechanisms of the parasite which might have evolved as a result of a long coexistence. Thus, the second approach is particularly inter- esting in tick-host systems where immunity seems not to be acquired during a natural exposure. Moreover, tick saliva which may manipulate local inflamma- tory and immune responses would also be avoided.

Rhipicephalus sanguineus is a dog ectoparasite and according to Pegram et al. (1987 ), it is the most widely distributed tick species in the world. It has been linked to many diseases (Hoogstraal, 1956; Sweatman, 1967; Parker and Wilson, 1979; Stephen and Achyutharao, 1980; P6ter et al., 1984; Fox and Sykes, 1985) and apparently does not induce resistance in its host, the dog, even after repeated feeding (Chabaud, 1950; Garin and Grabarev, 1972; Szab6 et al., 1993 ). Domes- tic dogs in Brazil have very heavy infestations of this tick and are a constant nuisance to the owners. Therefore, its control is most desirable. Moreover, the study of mechanisms involved in this parasitism will contribute to the under- standing of other host-parasite relationships if we consider the lack of resistance in the natural host against this tick species.

In the present study, as a first step towards the isolation of protective antigens, unfed adult extracts of the tick Rhipicephalus sanguineus was used for immuni- sation of dogs, hamsters and guinea pigs. Resistance inducement was tested ana- lysing various female tick performance parameters during a challenge infestation in each host.

2. Materials and methods

2.1. Ticks

An R. sanguineus tick colony was established to ensure a supply of unfed adult ticks for the experiments. Initially engorged females were collected from dogs of the Veterinary College Hospital in Jaboticabal, Brazil. Once identified, they were kept under a constant temperature of 28.9°C and a relative humidity of 80%. A continuous tick supply was then ensured by feeding each instar on tick-bite naive guinea pigs.

2.2. Hosts

Three animal species were used in these experiments; six mongrel dogs ( Canis familiares); ten hamsters (Mesocricetus auratus) and ten guinea pigs (Cavia cu- tleri). Dogs are the natural hosts ofR. sanguineus ticks and the other two species are commonly used laboratory animals. Every animal was tick-bite naive. For this purpose, dogs were bred in a tick-free room. The guinea pigs weighed about

G,H. Bechara et al. / Veterinary Parasitology 52 (I 994) 79-90 81

500 g and the hamsters about 120 g at the beginning of the experiments. All ro- dents were female. Two female and four male dogs of about 6 kg and 5 months of age were used. Water and food were given ad libitum.

2.3. Tick extract

Approximately 500 unfed adult Rhipicephalus sanguineus ticks were obtained from the colony. They were killed by immersion in liquid nitrogen, homogenised with a ground glass homogeniser in phosphate buffered saline (PBS, pH 7.4) and then sonicated three times for 10 s each time and once for 60 s (20 MHz). The extract was centrifuged at 4°C for 1 h at 12 000 ×g, the supernatant filtered through a 0.22/zm Millex-GV (Millipore) filter and stored at - 40°C until use. The protein concentration, determined according to Lowry et al. ( 1951 ), was 2.5 mg ml- 1

2.4. Immunisation of dogs

The immunisation procedure was similar for all three hosts: five hamsters, three (two male and one female) dogs and five guinea pigs were inoculated with the unfed adult extract (UAE) by subcutaneous route three times at 15 day intervals. Every hamster and guinea pig received 125 mcg of extract and 50 mcg of saponin (Quil A, Superfos Biosector A/S, Vedbaek, Denmark) as adjuvant diluted in PBS (pH 7.4) to a final volume of 1.0 ml per animal. Dogs received the same treat- ment but with 250 mcg of protein.

2.5. Challenge infestation

Fifteen days after the last inoculation all animals were submitted to a challenge infestation with unfed adult ticks. Tick-bite naive, control, and unvaccinated an- imals included five hamsters, three dogs (two males and one female) and five guinea pigs. The infestations consisted of four female and five male ticks per ro- dents and 25 females and 30 males per dog. Ticks were placed inside a feeding chamber consisting of a plastic tube (diameter 2.5 cm for rodents, 5 cm for dogs and height 3 cm), which the previous day had been glued with an atoxic and non- lesive preparation (Britannia Adhesives, Brentwood, UK) to the shaved back of the hosts. Elizabethan neck collars were also used on all animals to prevent grooming. In order to avoid the escape of ticks during experiments, hosts were kept in cages placed in trays surrounded by a gutter filled with water and oil. Daily observations were performed on some biological parameters of the female ticks.

2.6. Biological parameters

The following biological parameters, related to female tick feeding and repro- ductive performances, were observed during each infestation: engorged female weights (FW) and egg mass weights (EW), engorging, pre-oviposition and in-

82 G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90

cubation periods, larval hatchability rates (LH) and efficiency rates of female ticks in converting their food reservoir to eggs (ERCE) and larvae (ERCL). Fe- male weight was measured immediately after tick detachment. Egg mass was weighed 15 days after tick detachment as some preliminary observations dem- onstrated that there was no significant increase in this parameter after that period of time. The engorging period was assumed to be the timethat elapsed from the liberation of ticks on the hosts until their detachment partially or fully engorged. The pre-oviposition period was the time that elapsed from the detachment of the female tick until the beginning of oviposition. The incubation period was the time from the beginning of oviposition until the beginning of hatching of larvae. The larval hatching rate for each tick was obtained by the mean value of visual evaluation performed by three persons separately. The efficiency rates of conver- sion to eggs and to larvae were calculated as follows

E R c E = E W × 100 F W

ERCL = E W× LH FW

2. 7. Statistics

Data from biological parameters from each host species (challenge infesta- tion × first infestation) were analysed by analysis of variance. Means were com- pared by Tukey's test (P< 0.05 ). Comparisons of means were performed only intraspecifically between challenge infestations and infestation on control tick- bite naive hosts.

3. Results

The results of the various parameters are summarised in Figs. 1-3. Engorged female ticks (Fig. 1 (a)) fed on vaccinated hamsters and guinea pigs had a signif- icant (P<0.05) drop of 28 mg and 57 mg in their weights, respectively, when compared with ticks from control animals. However, ticks from dogs showed a significant increase of 13 mg under the same conditions. Egg mass weights (Fig. 1 (b)) showed a similar variation. There was a significant drop of 28 mg and 39 mg in the weights of the egg mass laid by ticks fed on vaccinated hamsters and guinea pigs, respectively, and an increase of 8 mg in the weight of the egg mass from ticks fed on vaccinated dogs when compared with ticks engorged on un- treated dogs.

The engorging period of female ticks (Fig. 2 (a)) fed on vaccinated hamsters, dogs and guinea pigs showed increases of 2.5 days, 0.3 days and 2 days, respec- tively, when compared with ticks fed on untreated hosts. All differences were considered statistically significant (P<0.05). The pre-oviposition periods did

G.H, Bechara et el. / Veterinary Parasitology 52 (1994) 79-90

we igh t (rag) (a)

'°° I ' i 140 ,

control- animals vaccinated animals

weight (rag) (b) IO0

80

6O

40

20

83

control animals vaccinated animals

I i ~ hamsters ~ dogs ~_~ guinea pigs II I I I I

Fig. 1. Parameters of R. sanguineus following infestation of naive or vaccinated hamsters, dogs and guinea pigs: (a) engorged female weight; (b) egg mass weight. Results are expressed as means +_ SEM.

84 G.H. Bechara ~'t al. / Veterinary Parasitology 52 (1994) 79-90

not vary as t hue h (Fig. 2 (b) ): there were increases of 0.4 days, 0.6 days and 0.2 days for ticks obtained from vaccinated hamsters, dogs and guinea pigs, respec- tively, when compared with ticks fed on controls. Only data from dogs were sta- tistically significant. The incubation periods of eggs (Fig. 2 (c)) from ticks fed on vaccinated animals showed less variation; there were respective decreases of 1.6 days and 1.0 days only in hamsters and guinea pigs compared with controls. Ticks from dogs displayed no difference between vaccinated and control groups.

There was no significant difference in larval hatchability rates (Fig. 3 (a)) be-

e n g o r g i n g ( d a y s ) (a) 1 2 ! . . . . . . . . . .

t t

10 ~ : ~ 1

6 4 i i !

2 ~

con t ro l an imals vaccinated animals

p r e - o v i p o s i t i o n (days) 5 ~ -

(b)

, r - - ~ - !

con t ro l animals vacc inated animals

G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90 85

25

20

15

10

incubat ion (days) (c)

control animals vaccinated animals

hamsters [ ~ dogs ~ guinea pigs I

Fig. 2. Parameters of R. sanguineus when infesting naive ox' vaccinated hamsters, dogs and guinea pigs: (a) engorging period; (b) pre-oviposition period; (c) incubation period. Results are expressed as means + SEM.

tween ticks obtained from vaccinated or control hosts. Slight increases of 1.8%, 4.6% and 2.3% were observed in ticks from treated hamsters, dogs a~ad guinea pigs, respectively, compared with ticks fed on controls..As for the efficiency rate of female ticks in converting their food reservoir to eggs (Fig. 3 (b) ), a significant drop of 11.9% and 25.7% in ticks from vaccinated hamsters and guinea pigs, re- spectively, was observed, compared with data from control hosts. Ticks from vac- cinated dogs showed a non-significant increase of 1.2%. The efficiency rate in conversion to larvae (Fig. 3 (c) ) was similar, with a non-significant decrease of 8.9% in ticks from vaccinated hamsters compared with ticks from control ham- sters; ticks from vaccinated guinea pigs displayed a significan~t (P~< 0.05 ) drop of 23.4% compared with ticks from the control group, and in vaccinated dogs there was a 4.0% increase compared with data from control dogs.

Local skin reactions at tick attachment sites, in every experimental condition, were generally slight in hamsters and dogs, consisting of some hyperaemia and oedema. Vaccinated guinea pigs, however, had a more intense reaction with a more prominent hyperaemia, oedema and exudation.

Two hamsters developed subcutaneous abscess following the third inoculation, and so were not challenged with tick infestation.

Tick recovery rates for the control and challenge infestations can be seen in Table 1. A lower recovery rate for dogs and guinea pigs during control infestation

86 G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90

was mainly related to accidental tick escape; during challenge infestation on vac- cinated guinea pigs some ticks engorged but did not oviposit and some, although attached, did not engorge, remaining this way until the feeding chamber was removed.

ha tchab i l i t y (%) (a) 100 ~-- i

' 4 i

80

60

40

20

control animals

I i

i

] ! i

vaccinated animals

e r c e (%) 70 . . . . .

6O I . . . . . . . . . .

5 0 -

i

4 0

0 control animals

(b)

vaccinated animals

G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90 87

ercl (%) (c) 7O

60

50

40

30

20

10

0

I

I

control animals vaccinated animals

[ ~ hamsters ~ dogs ~ guinea pigs I

Fig. 3. Parameters of R. sanguineus following infestation of naive or vaccinated hamsters, dogs and guinea pigs: (a) larval hatchability rate; (b) efficiency rate of female ticks in converting their food reservoir to eggs; (c) efficiency rate of female ticks in converting their food reservoir to larvae. Re- suits are expressed as means + SEM.

Table 1 Total numbers of Rhipicephalus sanguineus released and recovered from hamsters, dogs and guinea pigs

Dogs Hamsters Guinea-pigs

Control Vacc. Control Vacc. Control Vacc.

Released 87 75 21 15 20 26 Engorged a 71 70 21 14 16 24 Eggs laid 70 70 21 14 16 20 Recovered b (%) 80 93 100 93 80 77

aEngorged fully or partially. bTicks were considered as recovered if they attached, engorged and laid eggs.

4. Discussion

As described earlier (Chabaud, 1950; Garin and Grabarev, 1972 ) and accord- ing to our previous work (Szab6 et al., 1993 ), dogs do not develop resistance to the adult brown dog tick R. sanguineus following successive infestations. How-

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ever, hamsters and guinea pigs under the same conditions develop a marked re- sistance, characterised by a fall in engorged female and egg mass weights, pro- longed feeding period and lower efficiency rates of engorged females in converting their food reservoir to eggs and larvae.

Subsequently, we tried to induce resistance to R. sanguineus in dogs, hamsters and guinea pigs by inoculating them with unfed adult tick extract. This procedure is based on the presentation of tick antigens which usually are not involved in a natural host-parasite relationship. This would be a way of avoiding evasion mechanisms developed by the tick against the immune mechanisms of dogs dur- ing their long coexistence.

Even though vaccinated hamsters and guinea pigs developed a significant re- sistance against R. sanguineus, as ,clearly demonstrated by a decrease in engorged female and egg mass weights, decreased ERCE and ERCL in guinea pigs and pro- longed engorging period, dogs again did not display any observable resistance. Moreover, if data from the previous work (Szab6 et al., 1993) are compared, ~re~is.tance acclu~ired by vaccinated nadents seems to be less effective than resis- tzmce developed following successive intfestations. Experimental infestati@ns seem to be superior to vaccination in inducing resistance against R. sanguineus in guinea pigs and to sonae extent in hamsters, and that vaccination failed to induce resis- tance in dogs.

'These results; are not unexpected since a total tick extract was employed in these vaccinations and purified e~tracts ihave l~en shown to be more immuno- genie and even these are sometimes less effective in inducing resistance than pre- vious infestations (Morrison, 1989).

The dose of extract used (125 meg per rodent and 250 meg per dog) can be considered sufficient. Wikel et al. ( 1987 ) vaccinated guinea pigs successfully with gut extract of tlle tick Amblyomma americanum using 10 meg of extract per 250 g of host body weight and Obdebeeck et al. ( 1988 ) obtained good results inocu- lating gut and singanglion extracts of the tick Boophilus microplus into bovines using 550-1350 meg per host. However, the greater purification of these extracts must be taken into account.

Analysing the results obtained, it becomes clear that more purified tick extracts are needed to induce resistance in dogs against R. sanguineus. Further experi- mental steps should include extracts from isolated internal organs of the tick in vaccines as already done in other host-tick relationships (Brown et al., 1984; Ben-Yakir and Barker, 1987; Opdebeeck and Daly, 1990) and immunohisto- chemical techniques which could be utilised to find the most antigenic portions of these organs. Moreover, more purified extracts will probably avoid local reac- tions as observed in hamsters.

5. Conclusions

Crude adult R. sanguineus tick extract induced a very significant resistance in guinea pigs an, el a moderate but significant resistance in hamsters. However, R.

G.H. Bechara et al. / Veterinary Parasitology 52 (1994) 79-90 89

sanguineus did not induce any perceptible resistance in dogs against the same tick species. More purified antigens are needed for a more effective vaccination.

6. Acknowledgements

The present work was partially supported by the Conselho Nacional de Desen- volvimento Cientifico e Tecnol6gico (CNPq). The authors would like to thank Dr. Euclides Braga Malheiros for statistical assistance, Dr. Erik Lindblad, Super- fos Biosector A.S., Denmark, for the Quil-A saponin, a kind gift, the FundacSo de Apoio ~i Pesquisa no Estado de S~o Paulo-FAPESP (MPJS, LSM, PCSR) and the CNPq (GHB) for grants received. We are also most grateful to Edgar Homem and Narciso Batista Tel for helping with the animals.

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P6ter, O., Burgdorfer, W., Aeschlimann, A. and Chatelanat, P., 1984. Rickettsia conorii isolated from Rhipicephalus sanguineus introduced into Switzerland on a pet dog. Z. Parasitenkd., 70: 265-270.

Stephen, I.C. and Achyutharao, K.N., 1980. Natural occurrence of Coxiella burnetii in the brown dog tick Rhipicephalus sanguineus. Indian J. Med. Res., 71: 865-869.

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tance of dogs, hamsters and guinea pigs to infestations with adult ticks Rhipicephalus sanguineus (Acarina: Ixodidae). Vet. Parasitol. (submitted). From: Szab6, M.P.J., 1991. Aspectos da Imu- nopatologia Comparada em C$es, Hamsters e Cobaias a Carrapatos Rhipicephalus sanguineus (Latreille, 1806). MSc. dissertation, University of S~o Paulo.

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