Highlights of contemporary research on host immune responses to ticks

Veterinary Parasitology, 28 (1988) 321-334 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

321

Highlights of C o n t e m p o r a r y R e s e a r c h on Host I m m u n e R e s p o n s e s to Ticks* STEPHEN J. BROWN

Department of Biology, Loyola Marymount University, Los Angeles, CA 90045 (U.S.A.) (Accepted for publication 22 July 1987)

ABSTRACT

Brown, S.J., 1988. Highlights of contemporary research on host immune responses to ticks. Vet. Parasitol., 28: 321-334. Host immune responses to ticks have been known since the early part of this century. This research has emanated from throughout the world with the most detailed studies originating from Australia and the United States. A review of all the studies to date indicates a diverse tick species list consisting primarily of Ixodid ticks, but a few Argasid species have been examined. Typically, research on this topic during the first half of this century has utilized the bovine host, whereas research over the past 20 years has concentrated on the rodent host. The emphasis of this research has been to define host resistance in terms of behavioral and physiological changes in the host, accompanied by changes in the feeding and reproductive potential of the ticks. The primary objective of this research is to develop an innovative tick control strategy that will allow greater and safer control than that afforded by acaricides. This paper highlights the study of host immune responses to ticks over the century to date. However, owing to the great growth in the fields of immunology and molecular biology, the greatest gains have been made from 1970 to 1985. Therefore, the emphasis of this review is on research reported during the last 15 years.

INTRODUCTION

T h e ability o f v e r t e b r a t e s to acquire r e s i s t a n c e to feeding b y ticks is well d o c u m e n t e d . T h e first d e s c r i p t i o n of such a r e s p o n s e was in cattle by J o h n s t o n a n d B a n c r o f t (1918) (Table I). F r o m t h e t i m e o f this r e p o r t to t h e present, c o n s i d e r a b l e i n f o r m a t i o n has been g e n e r a t e d o n b r e e d susceptibility in relat i o n to r e s i s t a n c e of cattle a n d t h e heritability of such t r a i t s (Tables II a n d I I I ). I n addition, t h e adverse effects of this r e s p o n s e on t h e survival a n d reproductive c a p a c i t y o f ticks are well d o c u m e n t e d (review b y Willadsen, 1980). * This paper was presented at the President's Symposium entitled "Highlights of Contemporary Research on Tick Parasitism and Tick Borne Diseases" at the 1985 National Meeting of the American Society of Parasitologists, Athens, GA, U.S.A.

0304-4017/88/$03.50

© 1988 Elsevier Science Publishers B.V.

322 TABLE I Important events in the study of host resistance to ticks Year

Event

1908 Earliest report of bovine resistance to ticks (Hull; cited in Johnston and Bancroft, 1918) 1918 First published report of bovine resistance (Johnston and Bancroft) 1924 Concept of bovine innate resistance presented and demonstrated by cross-breeding studies (Lush) 1939 Guinea pig demonstrated to be a good model host and serum transfer of resistance reported (Trager) 1956 Theory of'Immunity or Skin Hypersensitivity' presented for the bovine (Riek) 1969 Histamine receptor antagonists shown to reduce bovine immune responses to ticks (Tatchell and Bennett) 1970 Passive immunization using salivary gland extracts demonstrated in the guinea pig (Garin and Grabarev) 1973 Basophils described at tick feeding sites in the guinea pig (Allen) 1976 Plasma transfer of resistance demonstrated in the bovine (Roberts and Kerr) 1977 Basophils described at tick feeding sites in the bovine (Allen et al.) Serum transfer of resistance in the rabbit (Brossard) 1979 Passive immunization using tick organs demonstrated in the bovine (Allen and Humphreys ) Langerhans cells in the guinea pig shown to bind tick antigens (Allen et al. ) 1982 Antibasophil serum treatment abolished guinea pig immunity to ticks (Brown et al. ) Guinea pig antitick antibody shown to be 7S IgG~ (Brown et al. ) Circulating basophils in the guinea pig shown to become sensitized with antitick antibody (Brossard et al.) 1984 Tick salivary gland-derived antigen is characterized (Brown et al. ) 1985 Guinea pig immunity requires the involvement of intact Fc receptors (Brown and Askenase ) Mast cell-deficient mice express normal resistance to Dermacentor variabilis (Den Hollander and Allen ) but fail to express resistance to Haemaphysalis longicornis ( Matsuda et al. )

TABLE II Listing of those animals classified as innately susceptible and innately resistant Susceptible

Resistant

European (Bos taurus)

Zebu (Bos indicus )

Shorthorn Hereford Jersey Holstein Friesian

Africander Sahiwal Sindhi Brahman

323 TABLE III Listing of proposed attributes of bovine innate resistance to ticks Attribute Short-hair coat Thin skin High sebaceous gland density High mast cell density Superficial hair follicles High number of circulating lymphocytes High protein diet

However, very little is known about the mediators and effectors of bovine immunity to ticks as well as the tick-derived substances responsible for the induction of immunity. The object of this brief review is to evaluate the contributions made to the field of host immunity to ticks, specifically those reported during the past 10 years, and to present these findings within the context of mediators, effectors, and inducers of immunity. Considerable progress towards understanding the mechanism of host immunity to ticks has been derived from studies using guinea pigs, and to a lesser extent rabbits, as model hosts for the bovine response. It is not assumed that the rodent's immunological and histological responses to tick feeding will be identical to that of the bovine. Rather, an understanding of the events surrounding tick rejection in the rodent will facilitate our understanding of similar events in the bovine. The justification for using rodent hosts is based upon cost, availability of high replication and ease of manipulation. Furthermore, the use of such hosts is not completely abnormal since the immature stages of most ixodid ticks use rodents as preferred hosts in the wild. GENERAL OBSERVATIONS ON VERTEBRATE IMMUNITY TO TICKS

From the time of the first report of host immunity in 1918 to approximately 1973, very little was known about this response in the bovine. It was well known that some cattle (African-bred Bos indicus) exhibited 'innate' resistance and acquired a level of immunity much greater than others (British-bred Bos taurus). In addition, resistant hosts responded to tick challenge with intensified grooming behavior, and tick attachment sites became marked by serous exudates which seemed to engulf the tick. More specific studies demonstrated that extracts of whole ticks injected into tick-sensitized hosts elicited strong macroscopic skin reactions (Riek, 1956) and plasma from animals expressing resistance contained anti-tick antibodies as shown by passive transfer (Roberts and Kerr, 1976), suggesting the presence of an immune-mediated response.

324

Very little was demonstrated with regard to the study of rodent immunity to ticks during the same time period. Trager {1939) established the importance of the guinea pig as a model host and demonstrated the ability of serum from animals expressing resistance to ticks to confer protection to naive animals. Other investigators examined the immune response in similar ways, but advances into the mechanism of host resistance was not achieved until 1969 with the publication by Tatchell and Bennett describing a reduction in bovine immunity to Boophilus microplus with the administration of histamine receptor antagonists. This study was followed 4 years later by a report of Allen (1973) who described the presence of basophils at Dermacentor andersoni feeding sites in resistant guinea pigs. The observed cutaneous basophilia was significant for two reasons: (1) this cell is not normally found in the tissue except during immunologic mediation, as shown years earlier {Richerson et al., 1969); (2) this cell contains vasoactive amines {whose activity is minimized or reduced by histamine receptor antagonists) like the mast cell and probably plays an important role in maintaining vasodilation, thereby facilitating immigration of potentially important effector and regulatory cells to the site of tick feeding. Subsequent to this, the published thesis work of Bagnall ( 1975 ), using Ixodes holocyclus and guinea pigs and rats, made a significant contribution to the field through comprehensive histopathology, cell and serum transfers, passive cutaneous anaphylaxis assays for the detection of antibody and immunization studies using crude extracts from ticks. The result of these two works was to establish the basis for subsequent studies aimed at elucidating the effectors oi immunity. The following discussion will serve to illustrate the importance oI these early findings to the present-day experiments aimed at defining the immunological regulation of host resistance to ticks. HISTOPATHOLOGICAL RESPONSES TO TICK FEEDING

The cutaneous cellular response at tick feeding sites in naive animals is characterized primarily by neutrophil infiltration with a weaker but marked eosinophil response {Table IV). The pathology is restricted to the immediate feeding site with the formation of a dermal feeding cavity distal to the mouth-parts that becomes hemorrhagic as feeding progresses. In addition, the vessels in the lower dermis are characterized by perivascular cuffing by neutrophils. Later in the feeding period, basophils begin to accumulate at the epidermal-dermal border but exhibit little degranulation. During this primary or sensitizing infestation, very little tissue destruction is seen. However, hosts previously exposed to ticks exhibit considerable pathology upon challenge. Macroscopically, reactions by tick-sensitized hosts to tick attachment and feeding are characterized by erythema. Microscopically, feeding sites are marked by intra-epidermal vesicles packed with basophils associated with an intense leukocytic cellular response adjacent to the tick's hypostome consisting pri-

325 T A B L E IV Twenty-four-h cutaneous dermal cellular responses (0.032 mm 2 area) at tick feeding sites in naive

and sensitizedguinea pigs Tick species ~

Amblyomma americanum

Primary response Basophils

Eosinophils

0

3+-12

0

0

0

Secondary response Neutrophils

Basophils

Eosinophils

Neutrophils

ND 4

113_+ 2

42_+ 9

ND

7+- 7

53+-25

3+- 1

9+2

3+1

12___ 3

50+ 5

16+ 4

6_+2

0

2_+1

ND

19_+ 6

15_+ 5

ND

3 -+13

1+1

ND

8 +_ 2

18+_ 3

ND

1 -+1

5-+1

18_+11

112_+52

11+ 3

4_+2

7 _+1

7+-4

5+ 4

249+52

75-+14

4+-3

(Brown and Askenase, 1981 )

Ixodes holocyclus (Brown et al., 1984)

Rhipicephalus appendiculatus (Brown et al., 1983)

Rhipicephalus sanguineus (Brown and Askenase, 1981 )

Dermacentor andersoni (Allen, 1973)

Ornithodoros tartakovskyi (McLaren et al., 1983 )

Ornithodoros parkeri (Johnston and Brown, 1985)

~Immature ticks used in each case. 2Mean +_s.e. :~Adjusted to 0.032 mm 2 area. 4ND = not determined.

marily of basophils (Table IV). Interestingly, basophils are not normal residents of the tissue and comprise only 1% of circulating leukocytes in most vertebrates (Schermer, 1967; Bloom and Fawcett, 1975) and increase by as much as 500% in tick-parasitized hosts (Gordon and Allen, 1979; Brown and Askenase, 1982). These cells accumulate in the tissue in response to specific immunologic recruitment involving T-cells and antibodies (Askenase, 1977). In addition, serum from hyperexposed guinea pigs that are expressing resistance contains a factor that stimulates basophil differentiation when incubated with bone-marrow cultures (Denburg et al., 1984). To date, the tick appears to be the most potent inducer of cutaneous basophilia, a form of delayed-type hypersensitivity. A N T I B O D Y AND C E L L - M E D I A T E D R E S P O N S E TO T I C K F E E D I N G

The presence of anti-tick antibodies in guinea pigs and rabbits expressing resistance to ticks is well documented and based upon in vitro assays (Kohler et al., 1967; Fujisaki, 1978) and in vivo assays by skin tests (Boese, 1974;

326 TABLE V Passive transfer (i.v.) of resistance to ticks with serum from guinea pigs expressing resistance Tick species

Stage ~ Serum transfer

Author

ml/100 g2 Rejection (%)

Boophilus microplus Amblyomma americanum Amblyomma americanum Rhipicephalus sanguineus Rhipicephalusappendiculatus Ixodes holocyclus Dermacentor andersoni Dermacentorvariabilis

A :~ L A L L L L

4.0 1.3 ~ 1.3 1.3 0.4 2.0 1.5 (ip) '~ 1.5(ip)

50 29 27 20 92 33 0 10

Roberts and Kerr, 1976 Brown and Askenase, 198 l Brown, 1982 Brown and Askenase, 1981 Askenase et al., 1982 Askenase et al., 1982 Wikel and Allen, 1976 Trager, 1939

L = larva, N = nymph, A = adult. eSerum volume transferred per 100 g host bodyweight. :~Bovine host. 4Activity was shown to be due to 7S IgGt antibody (Brown et al., 1982b ). "'Serum transferred at 2.5 ml/100 g i.v. resulted in 72% rejection {Brown, unpublished data, 1985 ).

McGowan et al., 1979) and, most importantly, by passive transfer (Table V). Furthermore, the antibody involved in both rabbit and guinea pig immunity is of the IgG class as demonstrated in vitro (Fujisaki, 1978) and in vivo after purification of the IgG i subclass (Brown et al., 1982b ). Similarly, the involvement of cell-mediated immunity in rodent immunity to ticks has also been demonstrated as shown by the ability of cells (lymph node derived or peritoneal exudates) from resistant animals to confer immunity to naive animals (Table VI ). Therefore, rodent immunity to ticks involves both antibody and cell-mediated immunity. Such a conclusion was suggested by earlier studies using the immunosuppressant drugs methotrexate (Allen, 1973) and cyclophosphamide (Wikel and Allen, 1976) that act upon T-cell and B-cell populations. However, selective depletion with these drugs is difficult to control because of their ability to act upon both arms of the immune system depending upon the dose. The passive transfer of either immune serum or immune cells resulted in the ability of recipient hosts to express significant immunity but not to the level exhibited by the donor host. Furthermore, cell transfer conferred a greater immunity than did serum, and both transferred together conferred a level of immunity only slightly greater than cells alone (Bagnall, 1975). The transfer of serum and cells also resulted in a cutaneous basophil response at tick feeding sites that temporally is comparable to actively sentisized and challenged animals {Brown and Askenase, 1981; Brown et al., 1984) (Table VII). This finding demonstrates the association between immune mediation-basophil accumulation in the tissue and host immunity to ticks. Serum conferred a

327 TABLE VI Passive transfer (i.v.) of resistance to ticks with serum from guinea pigs expressing resistance Stage ~ Cell transfer

Tick species

Author

Cells/100g2 Rejection (To)

Amblyomma americanum L Amblyomma americanum A Rhipicephalus sanguineus L Rhipicephalusappendiculatus L Ixodes holocyclus L Dermacentor andersoni L

9 × 106 9 X 10 7 9 × 10s 8)<107 8)< 107 2)< 107

Brown and Askenase, 1981 Brown, 1982 Brown and Askenase, 1981 Askenase et al., 1982 Askenase et al., 1982 Wikel and Allen, 1976

87% 35% 39% 91% 48% 25%

L = l a r v a e , N = n y m p h , A = adult. 2Cells transferred per 100 g of host bodyweight.

T A B L E VII Cutaneous cellular responses at tick feeding sites (0.032 mm 2 area) in guinea pig recipients of cells and serum from animals expressing resistance Tick system

Tick rejection (%) Basophils Eosinophils Mononuclears

A mblyomma americanum

Ixodes holocyclus

Rhipicephalus appendiculatus

Cell

Serum

Cell

Serum

Serum

61 75+7 9_+3 24 -+7

29 11+3 3_+ 1 32 _+7

48 96+8 14+2 25 _+4

33 43+11 3-+ 1 26_+ 4

90 26+4 6-+3 3 _+2

weaker level of protection than cells, and serum recipients expressed a level of cutaneous basophilia that is 15-45% of the level of cell recipients. These findings demonstrate further the importance of the basophil to rodent immunity to ticks. POTENTIAL EFFECTORS OF IMMUNITY

The definitive study that established the significance of cutaneous basophilia in the resistance response to ticks was demonstrated recently with the use ofpolyclonal rabbit anti-guinea pig basophil serum (Brown et al., 1982a). Ticksensitized animals treated with anti-basophil serum prior to challenge failed to express immunity. These animals were depleted of bone marrow, blood and tissue basophils. In addition, animals treated with rabbit anti-guinea pig eos-

328

inophil serum expressed reduced but significant immunity. These findings demonstrated the primary role of basophils in immunity to ticks with a secondary role for eosinophils, a relationship that is described as cooperative since eosinophil presence is in part basophil dependent (Ho et al., 1979 ). Tick-sensitized hosts respond to tick challenge within minutes to hours, manifested by intensified grooming behavior (Brown and Askenase, 1981). Pruritis is most likely mast-cell-dependent resulting from discharge of vasoactive amines, primarily histamine. Mast cells are common at tick attachment sites, and it has been proposed that bovine immunity to ticks is the result of a mast-cell-dependent eosinophil hypersensitivity (Riek, 1956; Schleger et al., 1976). However, mast cells comprise a very small proportion of the leukocyte population at tick feeding sites in bovines and guinea pigs and do not show any increase in density over time. However, the importance of the mast cell in immunity to ticks may reside in its ability to interact with tick antigen via surface-bound anti-tick antibody early during tick attachment acting as the initiator of the early phase of delayed-type hypersensitivity. This early phase activity by mast cells has been shown to be susceptible to a T-cell factor in the mouse (Askenase et al., 1983). Mast cell-deficient mice (WW v) have been shown to express normal levels of resistance to Dermacentor variabilis (Den Hollander and Allen, 1985) whereas WW v mice fail to express resistance to Haemaphysalis longicornis (Matsuda et al., 1985). The basophil, which also contains vasoactive amines, arrives in the tissue within hours of challenge, and increases to about 80% of the cellular density at tick feeding sites. The importance of this cell in rodent immunity to ticks has already been described so it is likely that basophil-derived mediators are crucial to this response. Several studies to date have demonstrated this to be true and found that ( 1 ) resistant animals had twice the level of tissue histamine than susceptible animals (Willadsen et al., 1979; Wikel, 1982), (2) histamine injected into the skin at tick attachment sites resulted in tick withdrawal or death (Kemp and Bourne, 1980), and histamine added to artificial media resulted in cessation of tick feeding (Paine et al., 1983 ), and (3) animals treated with histamine receptor antagonists expressed reduced skin reactivity and reduced resistance (Tatchell and Bennett, 1969; Bagnall, 1975; Brossard, 1982; Wikel, 1982). However, animals treated with antihistamines still allowed tick feeding, suggesting that either suboptimal doses of the drug were given or that histamine was of secondary importance. In a recent study, actively and passively sensitized animals treated with H-1 and H-2 histamine receptor antagonists expressed normal levels of immunity to A mblyomma americanum (Brown and Askenase, 1985b). These findings led the authors to believe that basophilderived histamine was of secondary importance in host immunity to this tick. However, they did demonstrate that a basophil-derived mediator was important and that there were two phases during tick feeding where ticks were sus-

329 ceptible to this/these mediator (s): 6 and 24 h after attachment coinciding with the attachment and induction of the fast feeding phases. Basophil-filled epidermal vesicles at tick attachment sites in animals expressing resistance are reminiscent of those occurring in contact hypersensitivity responses. These vesicles may develop in response to soluble or solidified (cement material) salivary gland secretions after processing and presentation by local antigen-presenting cells such as macrophages but, more importantly, Langerhans cells. These cells are Ia ÷ cells of the epidermis that bear receptors for Fc and C3. Langerhans cells are thought to act as antigen-presenting cells in the induction of contact hypersensitivity reactions (Ptak et al., 1980) and have been shown to bind tick salivary gland secretions by indirect immunofluorescence (Allen et al., 1979). There is evidence to suggest that tick-sensitized guinea pigs depleted of Langerhans cells fail to express resistance. Based upon this information, it appears that Langerhans cells take up and present tick antigens to sensitized T-cells which then secrete chemotactic factors that recruit circulating basophils, bound with antitick antibody, to infiltrate the local site and effect the immune resistance response. Basophils have recently been shown'to passively acquire anti-tick antibody in the circulation (Brossard et al., 1982 ). The mast cell can also carry surface-bound antibody, and it probably acts as the first line of defense inducing vasodilation by releasing vasoactive factors. This process facilitates cell immigration and is most likely maintained by the presence of basophils after mast cells have been used up. What is missing in this scheme is the inducer or antigen secreted by the tick during feeding. ANTIGEN INDUCERS OF IMMUNITY

Evidence supporting the opinion that the antigen (s) responsible for the induction and elicitation of host immune response to ticks is salivary glandderived is strong and based upon studiesusing guinea pigs (Wikel, 1981; Brown et al., 1984) and rabbits (Garin and Grabarev, 1972). CA review of immunization studies to date is listed in Table VIII). However, few reports have been published describing the characterization of tick products capable of inducing host immunity or elicitingskin responses in tick-sensitized animals (Table IX). Strong immediate skin responses were evoked in cattle by the injection of a protein from Boophilus microplusdescribed as an esterase with a molecular weight of 30 kDa (Geczy et al.,1971) and 60 kDa (Willadsen and Williams, 1976). In addition, antibodies against Boophilus microplus-derived phosphomonoesterases were found in tick-resistant cattle (Reich and Zorzopulos, 1980 ); this enzyme is associated with parasite-absorptive surfaces such as the gut. Recently, serum from guinea pigs expressing resistance to Amblyomma americanum was used to identify a single protein from the salivary gland secretions of this tick estimated to be 20 kDa (Brown et al., 1984), but no enzymatic activity was reported. Furthermore, immunoaffinity purified sal-

330 T A B L E VIII S u m m a r y of studies designed to test the efficacy of adult tick-derived substances to passively immunize animals against tick feeding Tick species

Stage ~ Extract preparation 2

Host '~

Result

Author

L A

WB WB, M G

GP Rat

+/+

Trager, 1939 Ackerman et al., 1980

A

WB

Rabbit

+

McGowan et al., 1980

A L

WB SGA

Bovine GP

+ +

McGowan et al., 1981 Brown et al., 1984

L

WB, MG RO SGA

Bovine GP GP

+ + +

Allen a n d Humphreys, 1979 Allen a n d Humphreys, 1979 Wikel, 1981

L,A

SGA

Rabbit GP

+/+

Garin a n d Grabarev, 1972 G a r i n and Grabarev, 1972

Dermacentor variabilis

A mblyomma maculatum A mblyomma americanum

Dermacentor andersoni

Rhipicephalus sanguineus

L = larva, N = n y m p h , A = adult. 2WB = whole body; M G = midgut; SGA = salivary gland; RO = reproductive organs. :~Guinea pig. T A B L E IX S u m m a r y of antigen characterization studies Tick species

Reported molecular weight (Da)

Biological activity

Assay used to detect antigenicity

Author

Boophilus microplus Boophihas microplu.s Boophilus mieroplus

30 0001 60 0 0 0 2 ND "~ 20 000

Skin test Skin test Neutralization by bovine AB Skin test, immunization, SDS/page of purified AG

Geczy et al., 1971 Willadsen and Williams, 1976 Reich and 7,orzopulos, 1980

Amblyomma americanum

Esterase Esterase Phosphomonoesterase ND

Brown et al., 1984

~Contained three isoenzymes. 2Contained two subunita. :~ND = not determined.

ivary gland antigen containing the 20 kDa species immunizes guinea pigs against feeding by Amblyomma americanum (Brown and Askenase, 1986). From this list of only a few studies to date, it is apparent that further work

331 is n e e d e d in t h e a r e a o f a n t i g e n c h a r a c t e r i z a t i o n . F o r t u n a t e l y , t h e r e c o g n i t i o n of t h e e c o n o m i c b e n e f i t in c o n t r o l l i n g t i c k s and, t h e r e f o r e , t i c k - b o r n e p a t h o gens is m o r e a p p a r e n t t h a n ever. T h i s r e a l i z a t i o n is leading m a n y i n v e s t i g a t o r s to a t t e m p t to isolate t h e crucial a n t i g e n (s) for e v e n t u a l v a c c i n e p r o d u c t i o n . SUMMARY

In s u m m a r y , h o s t - a c q u i r e d r e s i s t a n c e to t i c k s is i m m u n e m e d i a t e d , c h a r a c t e r i z e d b y c u t a n e o u s b a s o p h i l ( a n d e o s i n o p h i l ) i n f i l t r a t e s , r e s u l t s in signific a n t r e d u c t i o n in t i c k feeding a n d r e p r o d u c t i v e p o t e n t i a l a n d is i n d u c e d a n d elicited b y s a l i v a r y g l a n d - d e r i v e d s u b s t a n c e s . H o w e v e r , t h e site o f m o d e of a c t i o n b y t h e e f f e c t o r s a n d t h e i r p r o d u c t s in t h e tick is n o t k n o w n .

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