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IMMUNE RESISTANCE TO RHIPICEPHALUS SANGUINEUS IN DOGS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Sathaporn Jittapalapong, D.V.M.
The Ohio State University
1999
Dissertation Committee: Approved by
Professor John C. Gordon, Adviser Adviser Professor Roger W. Stich, Co-Adviser
Professor Lawrence E. Mathes Co-Adviser
Professor Daral J. Jackwood Veterinary Preventive Medicine Program UMI Number: 9941352
Copyright 1999 by Jittapalapong, Sathaporn
AH rights reserved.
UMI Microform 9941352 Copyright 1999, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
Rhipicephalus sanguineus is a cosmopolitan tick parasite of dogs that
also transmits several protozoal and rickettsial pathogens. Host Immunity to ticks would be an alternative to acarlcldes for the control of ticks and tickborne diseases, but the ability of dogs to acquire resistance against R. sanguineus has been questioned. The purpose of this study was to ascertain the effects of
Immunity Induced In dogs with tick salivary gland (TSG) or mIdgut (TMG) antigens compared to naturally acquired host resistance Induced by repeated
Infestations. Parameters of tick feeding and fecundity were then determined for subsequently challenge fed female ticks. Female ticks fed on dogs
Immunized with TSG had the lowest engorgement weight of all three groups.
A delayed feeding period and a decreased number of engorged females
Indicated resistance In the TSG-lmmunlzed group. Reduced fecundity was
Indicated In ticks fed on dogs Immunized with TMG by prolonged periods of pre-ovlposltlon, oviposltlon, and egg Incubation In addition to reduced egg production and viability, compared to that of ticks fed on naïve or TSG- lmmunlzed dogs. ELISA results Indicated cross-reactlvlty between sera collected from dogs Immunized with either TSG or TMG for either antigen preparation. Western blot analysis demonstrated nine bands of salivary gland
antigens, ranging from 10 kDa to 100 kDa, which were recognized by either
anti-TSG or TMG antibodies. Seven protein bands, at 450, 250, 210, 130, 125,
105, and 38 kDa, were uniquely recognized by sera from the TSG-immunized
dogs. Immunoblotting results indicated that antibodies specific for TSG might
be associated with lowered female body weight at engorgement, an important
feeding performance parameter that directly effects tick fecundity. At least nine
protein bands, ranging from 13 to 210 kDa, found in midgut, reproductive
organ and egg antigens were uniquely recognized by sera from TMG-
immunized dogs that suppressed tick fecundity. This work represents the first
essential step to characterize tick antigens that are associated with artificial O induction of reduced tick performance. Immunization of purified antigens associated with reduced tick feeding and fecundity performances may lead to greater resistance against R. sanguineus infestation in dogs.
I l l Dedicated to PHO and SOMPORN JITTAPALAPONG
IV ACKNOWLEDGMENTS
I would like to express most of my gratitude and appreciation to two persons, Dr. Omar O. Barriga (a former academic advisor), who give me inspiration and intellectual support and Dr. Roger W. Stich for encouragement and enthusiasm which made this dissertation possible and helped support me in the last portion of this work.
1 also thank Dr. John C. Gordon, Dr. Cliff Monahan, Dr. Lawrence E.
Matthes, Dr. Thomas E. Wittum, Dr. Daral J. Jackwood and Dr. Glen
Needham for their valuable suggestions. I am grateful to Catherine A. Bremer for the excellent technical assistance as well as staff and faculties of
Veterinary Preventive Medicine for their helps and supports.
This research was funded by the Morris Animal Foundation, the
Graduate School Alumni Research Award (The Ohio State University) and
Kasetsart University Research and Development Institute of Thailand.
Lastly, I wish to thank. Royal Thai Government to support for the scholarship, and Dr. Weeraphol Jansawan, the chief of Parasitology
Department, Kasetsart University for their concern with my studies.
I would like to dedicate this work to my father, mother and family with my thanks for their support throughout my education. VITA
February 14, 1959. Born-Nonthaburi, Thailand
1981...... B.Sc. Kasetsart University, Thailand
1983...... D.V.M. Kasetsart University, Thailand
1983-1988...... Instructor,
Faculty of Veterinary Medicine,
Kasetsart University, Thailand
1988-1993...... Assistant Professor,
Faculty of Veterinary Medicine,
Kasetsart University, Thailand
1993-Present ...... Graduate student.
The Ohio State University
1998-1999...... Graduate Teaching and Research
Assistant, Department of Veterinary
Preventive Medicine,
The Ohio State University
VI PUBLICATIONS
1. Jittapalapong, S.1981. Coumarin poisoning in Dogs. Journal of Thai Veterinary Practitioner 3 (4): 263-273.
2. Jittapalapong, S.,Jansawan, W., Sriworanat, P., and Daengsupha, P. 1986. Infection Rate of Ascaris iumbricoides in pigs. Journal of Kasetsart University Veterinary Medicine 7(3): 144-153.
3. Chantharayotha, 0., Khuphophan, S., Jansawan, W., Sriworanat, P., and Jittapalapong, S., 1986.Intestinal Parasites Causing Piglet Diarrhea in Four Provinces in Thailand. The Annual Report of Kasetsart University. 121.
4. Jittapalapong, S.,Jansawan, W., and Pinyopummin, T. 1987. Epidemiological Study of Dairy Cow in Nongpho. Journal of Kasetsart University Veterinary Medicine 8 (1): 21-29.
5. Jittapalapong, S.,Jansawan, W., and Pinyopummin, T. 1987. Epidemiological Study of Calves in Nongpho. Journal of Kasetsart University Veterinary Medicine 8 (1): 124-132.
6. Sunthrasamai, P., Warrell, M. J., Warrell, D. A., Chanthavanich, P., Looareesuwan, S., Supaphchana, A., Phanuphak, P., Jittapalapong, S., Yager, P. A., and Baer, G. M. 1987. Early Antibody Responses to Rabies Post-exposure Vaccine Regimens. American Joumai of Tropical Medicine and Hygiene 36{^): 160-165.
7. Jittapalapong, S.,Jansawan, W., Sriworanat, P., and Daengsupha, P. 1990. Survey Natural and Experimental Reservoir Host of Capiiiaria phiiippinensis in Pet and Wild Animals. Joumai of KU Veterinary Hospital 3(1): 69-77.
8. Jittapalapong, S.1990. Epidemiological Study of HeartWorm Disease in Dogs in Bangkok and Vicinity. Joumai of Thai Veterinary Practitioner^2{4): 265-277.
9. Jittapalapong, S.,and Tipsawek, S. 1991. Epidemiological Survey of Blood Protozoa and Rickettsia in Dogs in Bangkok and Vicinity. Kasetsart University Joumai25{^): 75-82.
10. Jittapalapong, S.,and Jansawan, W. 1993. Preliminary Study of Blood Parasites in Cats in Bangkok. Kasetsart University Journal 27(3): 330-335.
VII 11. Jansawan, W., Jittapalapong, S.,and Jantharat, N. 1993. Effect of Stemona collinsae Extract against Cattle Ticks {Boophilus microplus). Kasetsart University Joumai 27(3): 336-340.
12. Jittapalapong, S.,Intharaksa, Y., Jantharat, N., and Phatthanathanang, K. 1993. Epidemiological Survey of Intestinal Parasites of Calves in the Northeastern Provinces of Thailand. Kasetsart University Journal 27(4): 469-473.
13. Jittapalapong, S.,Stich, R. W., Gordon, J. C., Wittum, T. E., and Barriga, O. O. 1999. Reduced Feeding and Fecundity Performance of Rhipicephalus sanguineus (Acari: Ixodidae) by Salivary Gland or Midgut Immunizations, and Repeated Infestations in Dogs (Submitted)
14. Jittapalapong, S.,Stich, R. W., Gordon, J. C., Bremer C. A, and Barriga, O. O. 1999. Humoral Immune Response in Dogs previously Immunized with Salivary gland. Midgut or Repeated Infestations with Rhipicephalus sanguineus. (New York Academy of Science:In press)
FIELDS OF STUDY
Major field: Veterinary Preventive Medicine
VIU TABLE OF CONTENTS
Pages
Abstract...... li
Dedication...... iv
Acknowledgments...... v
V ita...... vi
List of Tables...... Xii
List of Figures...... Xiv
Chapters:
1. Introduction...... 1
2. Feeding and fecundity performance ofRhipicephalus sanguineus
(acari: Ixodidae) fed on dogs after multiple infestations and
immunization with tick salivary glands or midgut tissues...... 44
2.1 Introduction ...... 44
2.2 Materials and Methods ...... 46
2.3 Results ...... 50
2.4 Discussion ...... 54
2.5 References ...... 60
ix 3. Humoral immune response in dogs previously immunized with
salivary gland, midgut, or repeated infestations with
Rhipicephalus sanguineus...... 72
3.1. Introduction ...... 72
3.2. Materials and Methods ...... 75
3.3. Results...... 78
3.4. Discussion ...... 80
3.5 References ...... 83
4. Salivary glands protein profilesof Rhipicephalus sanguineus and
other tick species during infestation recognized by resistant sera of
exposure dogs with tick tissue immunization or repeated infestations
...... 89
4.1. Introduction ...... 89
4.2. Materials and Methods ...... 92
4.3. Results ...... 95
4.4. Discussion ...... 99
4.5. References ...... 102
5. Protein profiles of different stages and internal tissues of
Rhipicephalus sanguineus antigens recognized by resistant sera of
dogs previously immunized or repeated infestations...... 116
4.6. Introduction ...... 116
4.7. Materials and Methods...... 119
X 4.8. Results ...... 122
4.9. Discussion ...... 125
4.10. References ...... 132
6. Synopsis ...... 143
Bibliography...... 149
XI LIST OF TABLES
Tables Pages
3.1: Feeding and fecundity performance of female Rhipicephalus sanguineus ticks fed either on dogs that had been immunized with tick midgut (TMG) or salivary gland (TSG) and challenge infestations, or repeatedly infested (INF) with adult male and female of R. sanguineus. Results are shown as Least Square (LS) Means ± Standard Error (SE) superscript indicate a significant difference between values within each parameter as determined by Tukey-Kremer method Least Square Mean with statistical difference (p value < 0.05) ...... 87
4.1: Molecular weight ranged between 100-450 kDa of salivary gland proteins at different infestation immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R sanguineus ...110
4.2: Molecular weight ranged between 7-95 kDa of salivary gland proteins at different infestations immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R. sanguineus...... I l l
4.3: Western blot results of female R. sanguineus salivary gland proteins (kDa) at different infestations recognized by sera of dogs that were previously repeated immunized by using salivary gland (TSG) or midgut (TMG) extract or multiple infested (INF) by adult males and females of R. sanguineus...... 112
4.4: Molecular weight ranged between 105-450 kDa of unfed salivary gland proteins of different tick species immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R. sanguineus ... 113
XII 4.5; Molecular weight ranged between 10-100 kDa of unfed salivary gland proteins of different tick species immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R. sanguineus ... 114
4.6: Western blot results of unfed salivary gland proteins (kDa) of different tick species recognized by sera of dog that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus...... 115
5.1: Western blot results of tick developmental stage proteins including egg mass, unfed larvae, fed larvae, and nymphs recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus...... 138
5.2: Western blot results of developmental stages of R. sanguineus recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen, or multiple infested (INF) by adult females and males of R. sanguineus...... 139
5.3: Molecular weight of tick tissue proteins ranged between 95-420 kDa recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus...... 140
5.4: Molecular weight of tick tissue proteins ranged between 7-85 kDa recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus...... 141
5.5: Western blot results of tick-tissue antigens recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus...... 142
■XIU LIST OF FIGURES
Figures Pages
2.1; Feeding performance parameters of R sangu/neas females during repeated infestations on dogs. Dogs were infested five times by 80 female and 40 male ticks per infestation at 21 d intervals. After engorgement, the detached females were immediately weighed and incubated separately to observe feeding performance parameters of each individual including the engorged number (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Results are shown as Least Square (LS) Means ± Standard Error; different letters in each panel indicate significantly different LS Means (P < 0.05)...... 66
2.2: Fecundity parameters off?, sanguineus females during repeated infestations on dogs. Dogs were infested, ticks collected and results are reported as described for Fig. 1. Fecundity parameters measured included the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre-oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by the weight of the egg mass produced by that same tick ...... 67
2.3: Feeding parameters of R. sanguineus females from all infestations fed on immunized or repeatedly infested dogs. In the immunized groups, dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The immunized dogs were subjected to two challenge infestations with 80 female and 40 male ticks per infestation, separated by a 21 d interval, one week after the final immunization. Dogs in the repeated infestation group were infested five times at 21 d intervals with 80 female and 40 male ticks per infestation. The bars in each panel represent the Least Square (LS) Mean ± Standard error of tick
x i v performance parameters from all infestations in the repeated infestation, midgut, and salivary gland immunized groups. Feeding performance parameters of each individual included the number engorged (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Different superscript letters represent significantly different (p < 0.05) measurements within each panel ...... 68
2.4: Fecundity parameters of R. sanguineus females from all infestations fed on dogs that were repeatedly infested or immunized with tick tissues. Feeding parameters of R. sangu/neus females fed on immunized or repeatedly infested dogs. Dogs were repeatedly infested five times or immunized three times with tick salivary glands or midgut followed by two challenge infestations. The bars in each panel represent the Least Square (LS) Mean ± Standard error of ticks from all infestations in the repeated infestation, TMG, and TSG groups. Fecundity performances measured included the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre-oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by weight of the egg mass-produced by that same female. Different superscript letters represent significantly different (p < 0.05) measurements within each panel ...... 69
2.5: Feeding parameters of R sangu/neus females from individual infestations of dogs immunized with tick tissues. Dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The dogs were subjected to two challenge infestations with 80 female and 40 male ticks per infestation, separated by a 21 d interval, one week after the final immunization. Values from the first two repeated infestations are included as controls. Engorged, detached females were immediately weighed and incubated separately to observe feeding performance parameters of each tick, parameters included the number engorged (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Closed and open bars represent the first and second challenge infestations, respectively. Results are shown as Least Square (LS) Means ± Standard Error; different
XV superscript letters in each panel indicate significantly different LS Means (p < 0.05)...... 70
2.6: Fecundity parameters of R. sanguineus females from individual infestations on dogs immunized with tick tissues. Dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The dogs were subjected to two challenge infestations, separated by a 21 d interval, with 80 female and 40 male ticks per infestation one week after the final immunization. Engorged, detached females were weighed and incubated separately to observe all fecundity performance parameters of each individual including the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre-oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by weight of the egg mass-produced by that female. Closed and open bars represent the first and second challenge infestations, respectively, for each panel displayed. Results are shown as Least Square (LS) Means ± Standard Error; different superscript letters in each panel indicate significantly different LS Means (p <0.05) ...... 71
3.1: Humoral responses of dogs previously immunized with tick salivary gland (TSG) or midgut (TMG) or repeatedly infested (INF) were tested by ELISA to egg mass (a), unfed larva (b), fed larva (c), nymph (d), midgut (e), and salivary gland (f) antigen. In TSG or TMG group, dogs were immunized three times and challenge twice infestation by 80 female and 40 male ticks per infestation at 21-day intervals. In the repeated infestation group, dogs were infested five times with the same number of ticks. The series of sera (x-axis) of dogs including pre-immune (1), after the first immunization or infestation (2). after the second immunization or infestation (3), after the third immunization or infestation (4), after the first challenge infestation or the fourth infestation (5), and after the second challenge infestation or the fifth infestation (6) were collected and performed ELISA and their results were measured at OD 405 (y-axis) ...... 88
4.1: Western blot results of male and female salivary gland protein profiles during the first infestation of R. sanguineus recognized by a-TSG (A), a- TMG (B), and a-repeated infestation (0) dog sera (lane 1= unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day fem ale) ...... 106 xvi 4.2: Western blot results of male and female salivary gland protein profiles during the second infestation of R. sanguineus of R. sanguineus recognized by a-TSG (A). a-TMG (B), and a-repeated infestation (C) dog sera (lane 1= unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day fem ale) ...... 107
4.3: Western blot results of male and female salivary gland protein profiles during the third infestation of R. sanguineus of R. sanguineus recognized by a-TSG (A), a-TMG (B). and a-repeated infestation (0) dog sera (lane 1= unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day female) ...... 108
4.4: Western blot results of salivary gland protein profiles of different tick species recognized by a-TSG (A) and a-TMG (B) sera of dogs (lane 1 = unfed male R. sanguineus] 2 = fed male R. sanguineus] 3 = unfed male A. americanum] 4 = unfed female A. americanum] 5 = unfed female A. cajennense] 6 = unfed female D. variabilis] 7 = unfedfemale R. sanguineus] 8 = fed female R. sanguineus] 9 = TSG vaccine antigen) 109
5.1: Western blot analysis of different stages and tissues of R. sanguineus immunoblotted with different anti-TSG-sera; after the last immunization (A), after the first challenge infestation (B), and after the second challenge infestation (C). (Lane 1=egg mass; 2 = unfed larvae; 3 = fed larvae; 4 = nymphs; 5 = midgut; 6 = reproductive; 7 = muscle; 8 = nerve, and 9 = salivary gland extract) ...... 135
5.2: Western blot analysis of different stages and tissues of R sanguineus immunoblotted with different anti-TMG-sera; after the last immunization (A), after the first challenge infestation (B), and after the second challenge infestation (C). (Lane 1 = nerve; 2 = muscle; 3 = reproductive; 4 = midgut; 5 = salivary gland; 6 = nymphs; 7 = fed larvae; 8=unfed larvae, and 9 = egg m ass) ...... 136
5.3: Western blot analysis of different stages and tissues of R sanguineus immunoblotted with different a-repeated infestation sera; after the third infestation (A); after the fourth infestation (B); and after the fifth infestation (C). (Lane 1 = nerve; 2 = muscle; 3 = reproductive; 4 = midgut; 5 = salivary gland; 6 = nymphs; 7 = fed larvae; 8 = unfed larvae, and 9 = egg m ass) ...... 137 xvii CHAPTER 1
INTRODUCTION
It is estimated that in the phylum Arthropoda, the class Insecta alone
embraces from 625,000 to 1,500,000 species comprising approximately 8
percent of all known animal species (Tatchell 1969). Through this tremendous
diversification, arthropod life can be found almost anywhere on earth, so the
contact of man and other animals with arthropods is virtually unavoidable.
Throughout the centuries man and animals have been plagued by arthropods which consume enormous quantities of food and which are responsible for
numerous pathologic conditions either by serving as vector of etiologic agents of disease or by being themselves the causative agents.
1.1. Identification and Classification
Ticks belong to the superfamily Ixodoidea, which is divided into two major families, Ixodidae and Argasidae. The family Ixodidae or a hard tick contains about 660 species (Technical Information Memorandum no26, 1998).
As adults, these ticks exhibit prominent sexual dimorphism: a sclerotized plate called the scutum covers the entire dorsum in males, but in females (and immatures) the scutum is reduced to a small shield behind the capitulum that
1 permits new tegument expansion during blood feeding. All ticks have a six
legged larval stage, one or more eight-legged nymphal stages, and an eight legged adult stage. Ixodid (hard) ticks have a single nymphal stage, while argasids (soft) ticks may have as many as eight. Hard ticks generally take one blood meal per stage; the adult female oviposits and dies following the engorgement. The male feeds little and dies following mating. Soft ticks feed intermittently and adult females may feed and oviposit several times
(Sonenshine, 1991).
Ticks have a wide range of host relationships. They are commonly classified as being one-host, two-host, and three-host ticks. One-host ticks complete all feeding and molting on a single animal. In two-host ticks, the molt from larva to nymph takes place on the host and the engorged nymph drops to the ground following feeding, where it molts to the adult stage. The adult must later find a second host. Three-host ticks leave their host after engorging at each life stage. These various life stages of ticks use a variety of species as hosts, increasing the number of potential sources of pathogens. Most ixodid ticks are three-host ticks. Most soft ticks feed on a number of different animals during their life cycle, with the adult feeding several times.
Rhipicephalus sanguineus is a member of the family Ixodidae, which is characterised by terminal mouth part and the presence of a tough, sclerotized plate on the dorsal body surface, the scutum; elsewhere on the body, the cuticle is characterised by innumerable tiny surface folds that penetrate only partially Into the cuticle (Sonenshine, 1991). This tick’s size Is small to
medium, as adults are usually less than 5mm long by 2mm wide with short,
broad palps. This specie lack ornamentation. The basis capltull Is distinctive,
with protruding, pointed lateral margins presenting a distinctly hexagonal
shape. The body In both sexes has eyes and festoons.
Rhipicephalus sanguineus, the ‘brown dog tick’. Is a cosmopolitan
Ixodid tick species that feeds on dogs during Immature and adult stages of
development (Cupp, 1991). This tick has been Implicated In the transmission
of several parasites that cause additional losses for pet owners. Including
Babesia canis, B. gibsoni, Ehrlichia cam's, and Hepatozoon cam's (Levine
1961; Ewing 1969; Senevlratna et al. 1973; Groves et al. 1975). This tick was
also reported as a vector of Anaplasma marginale (Parker, 1982), Coxiella
burnetii {Stephen et al. 1980), and Rickettsia conori (Injeyan et al. 1971).
Recent reports have shown that R. sanguineus occasionally parasitize
humans. Indicating a potential role for transmission of zoonotic pathogens as well (Goddard 1989; Carpenter et al. 1990; Guglielmone et al. 1991).
1.2. External and Internal Morphology
Mouth part
The mouthpart of arachnids (spiders, ticks, and mites) is different from the hematophagous Insects since the former does not have true mandibles.
Instead, the arachnids possess a pair of chelicerae (pincers) which serves for grasping the food source, and a highly developed sucking apparatus which serves for the intake of the liquid meal. The trauma caused by the mechanical
action of the mouthparts during the bite varies in magnitude depends both on the arthropod’s mode of feeding and on the host. In addition, the magnitude of the trauma is affected by the size of the arthropod and by the duration of feeding.
Salivary gland
In addition to the mechanical process of the bite, other processes are involved in the acquisition of the blood meal. One such important process is the injection of oral secretions into the host's tissue. Little is known about the mechanism which triggers the output of oral secretion of hematophagous arthropods; it would, however, be expected to be under nervous control similar to that inferred for some phytophagous arthropods (Day and Waterhouse,
1953). The composition and function of the oral secretion of hematophagous arthropods vary depending on the food source, diet, and feeding habits. The oral secretion of the slow feeding tick, Dermacentor andersoni, contains substances, presumably originating in the salivary glands, which harden into a latex-like material which moulds itself around the mouthparts of the tick and the skin of the host, thus enabling the tick to attach itself firmly to the host for a prolonged period (Gregson, 1960). Other compounds which undoubtedly play an important role in the feeding processes are the anticoagulants, which were demonstrated in the oral secretions of several hematophagous arthropods
(Ribeiro et al. 1985; Ribeiro & Spielman 1986). Although their presence is not universal, anticoaguiatory substances are particularly important when the arthropod feeds on a pool of blood rather than directly from the host's capillary. The oral secretions of hematophagous arthropods contain numerous other compounds including hemagglutinins, toxins, sugars, amino acids, peptides, proteins, phenolic compounds, and many others (Ribeiro 1987a, b;
Ribeiro 1989).
The substances in the oral secretions of arthropods have the tremendous importance of inducing local and systemic reactions of the host immunity. The response of the host to the bite of the arthropod varies, depending primarily on the nature of host or the arthropod's oral secretion. A host who may react strongly to the bite of one species may not react to bites of other species. Similarly, a given species of arthropod may elicit extreme responses in some individuals but not in others. In addition to the degree of intensity of responses, there also exist variations in the time of appearance and in the duration of the reactions following the bite. Thus, apart from the trauma inflicted on the host by the mouthparts during the process of feeding, reactions of the host to the bite may be attributed to substances present in the oral secretion.
The salivary glands of ticks serve as primary osmoregulatory organs by which ions and water are eliminated in the host during feeding (Kaufman &
Sauer, 1982), and salivary components maintain the intimate association between the parasite and host for days or even weeks (Allen, 1989; Ribeiro, 1989). Tick saliva contains many antihemostatic and antiinflammatory proteins
that facilitate blood feeding and repress host immune response to the tick
(Ribeiro, 1987). The salivary glands increase tremendously in size and protein content after the onset of feeding (McSwain et al. 1982). Other proteins present in unfed ticks increase in quantity during the later stages of tick feeding. Mating stimulates additional feeding and an increase in the amount of proteins (Sauer et al. 1986). In the brown dog tick, no report exists about the protein change during feeding in either sex. Thus to study the production of saliva it is essential to follow the sequence of changes in both sexes throughout feeding.
The paired grape-like salivary glands are situated laterally in the hemocoel and extend from close to the mouthparts to the spiracles. The structure of Ixodid salivary glands has been studied by several authors in the past twenty years (Balashov, 1968; Kirkland, 1971; Coons & Roshdy, 1973;
Megaw & Beadle, 1979; Fawcett et al. 1981a and b, and Krolak et al. 1982).
The glands contain two functionally distinct types of acini: one concerned with the elimination of excess fluid, the other with the secretion of granular materials. The secretory function of Ixodid salivary glands depends on the weight of the tick and the stage of feeding. Four types of cell morphology are found during development and feeding period of male and female adult ticks
(Sauer et al. 1995). (1) Acinus I (Type I alveoli) connecting directly to the main salivary duct
are present in larvae, nymph and adults. These cells are non-granular
and lack individual cell boundaries. These acini remain constant
morphologically through feeding (Gill and Walker 1984)
(2) Acinus II (Type II alveoli) located directly via a short lobular duct,
which surrounded a central lumen and opened via a cuticular valve into
a lobular duct. These acini cells contain at least six granular cell types
(a, b, and c1-c4). After several days of tick feeding, the morphology of
the alveolus undergoes a remarkable transformation without a change
in the number of cells. The nuclei and cytoplasm of most cells enlarge
during tick feeding, resulting in an overall increase in the mass of the
alveolus. Much of the granular material observed in the salivary gland
cells of unfed ticks and in the early stages of tick feeding, and is absent
during the final feeding stages. Type II alveoli are morphologically
similar to type III alveoli.
(3) Acinus III (Type III alveoli), are located on the peripheral and posterior regions of the gland, which contained three granular cell types
(d, e, and f), and are the most abundant in the salivary gland. During tick feeding, the cell membrane of each alveolus proliferate and the number of mitochondria increase. At the same time, the abluminal interstitial cells hypertrophy, with a parallel increase in mitochondria and plasma membranes, resulting in the formation of a basal labyrinth. The
7 large number of type III alveoli and the increase in alveolus size
suggest that the bulk of excreted fluid be transported through this
alveolus type during tick feeding.
(4) Acinus IV (Type IV alveoli), present only in males, developed from
clusters of undifferentiated nuclei in the glands of the pharate male.
These acinus cells have only one granular cell type (g). There is an
enormous increase in these granules toward the end of feeding. No
difference between glands from mated and un mated males was found
(Binnington, 1978). Male ticks that do not feed on the host have only
two types of alveoli, I and IV (Stone & Binnington, 1986).
The size, mass, and protein content of the salivary glands in adult
Ixodid females increase about 25-fold during feeding (McSwain et al. 1982;
Shipley et al. 1993). Tick feeding stimulates differential gene expression in the
salivary glands (Oaks et al.1991). The final phase of control over Ixodid female
salivary gland differentiation, the degeneration of the salivary glands, occurs
as the female attains her repleted weight and drops from the host. This
process is initiated by the release of tick salivary gland degeneration factor,
which is probably A 20-hydroxyecdysone (Harris & Kaufman, 1985). This
factor is released when the female reaches a mass 10 times greater than its
unfed mass (Kaufman, 1986; Lindsay & Kaufman, 1988). In mated females, degeneration is almost complete within four days following detachment from the host.
8 Ticks are slow feeding, temporary ectoparasites, which leave the host only to molt and to oviposit. Hard ticks are feeding generally lasts 3-5 days in adult females and 7 or more days in immature stages (Balashov, 1984). The body weight of females increases by more than 100 times (Balashov, 1976).
At every developmental stage of the life cycle, hard ticks feed only once, and the blood meal is sufficient for the molt to occur to the next stage. Females deposit, depending on the species, from 1,000-3,000 (Ixodes,
Haemaphysalis) to 10,000-20,000 eggs (Hyalomma, Ambiyomma) (Balashov,
1967). Most Ixodid are three host ticks.
Furthermore, salivary glands are a medium for the transmission of many types of disease organisms such as viruses, rickettsia (Anaplasma marginale, Cowdria ruminatum. Ehrlichia canis, and Haemobartonella cam's), protozoan (Babesia canis, B. bigemina, B. bovis, B. gibsoni and Hepatozoon canis), and spirochetes (Binnington & Kemp, 1980; Nuttall et al. 1994; Ribeiro
1987; 1989).
1.3. Tick-Host Association
Three host ticks have a wide variety of vertebrate host. Most Ixodid have preferential body attachment areas and are rare or absent elsewhere.
Thus, female /. ricinus usually attaches to cattle in the groin and i. persuicatus on the neck and dewlap. Dermacentorpictus attaches to cattle on the ears, near the tail tip, occipital fold, and on the back and sides of the neck.
Rhipicephaius turanicus usually attaches to sheep ears and occasionally to the inner side of the tail, groin, and other body parts sparsely covered by hair
and lacking abundant grease and sweat secretions. However, this tick
attaches to all body parts immediately after shearing. Irregular tick distribution
on the host body is associated with capitulum length and vertebrate skin
thickness, and the animal’s grooming ability (Arthur, 1962).
The effect of host grooming on parasite distribution on the host has
been most studied in small mammals. Kucheruk et al (1955, 1956) found larval
Dermacentor marginatus distributed regularly on the body of the hedgehog, as
this animal has lowered the ability to remove ticks by scratching or biting. In
contrast, on a common field mouse, 96% of larvae attached to the ears and
very few to other parts of the head, suggestion that the rodent can remove
ticks from most body parts.
Tick death may also result through active destruction by various host
behavior reactions and through internal changes of the host, which may hinder
normal tick feeding. Not all attached ticks successfully complete feeding and
detach from the host, despite the absence of mechanical damage. Great
differences are found among various host species as well as among individual
hosts of one species (Barriga et al. 1994)
Various degrees of specific innate resistance to Ixodid tick attack may
be associated with skin structure, but the basis of this resistance is more likely to be found in the specific biochemical and physiological characteristics of
10 each host species. Individual innate resistance to ticks is variable but may be
known as specie resistance.
1.4. Medical and Veterinary Importance
Apart from adverse psychological reactions to arthropods and the
consequences of such reactions, arthropods may be the direct cause of
pathologic conditions in man and other mammals by the following factors.
(1) Bites (e.g., mosquitoes, lice, mite, tick, etc.)
(2) Inoculation of toxins (e.g., venom of scorpions, spiders, bees, and
wasps)
(3) Invasion into the tissues ( e.g., certain mites and dipterous larvae)
(4) Contacts ( e.g., various stages of some Lepidoptera and blister
beetles)
(5) ingestion ( e.g., shrimp, lobsters, crayfish, and certain dipterous
larvae), and
(6) Inhalation (e.g., house dust mites and arthropod fragments).
The early discoveries that hematophagous arthropods serve as vectors of diseases such as malaria, typhus, plague, sleeping sickness, gall sickness,
red water fever. Rocky mountain spotted fever, and yellow fever greatly intensified research on their roles in the transmission of many other diseases affecting humans and animals. Nevertheless, apart from disease transmission, the manner in which the hematophagous arthropods obtain their blood meal more often than not triggers a reaction by the host. This reaction may not only
11 be unpleasant; it may actually result in severe manifestations ranging from
pruritus and generalised urticaria to systemic anaphylaxis and death. Actually,
it is of great importance, both from the standing point of the host-parasite
relationship and from the standing point of its possible role in disease
transmission. By virtue of dependence on the host for survival, the degree of
reactivity of the host to the bite of the arthropod may, to a certain extent, play a
role in the host-parasite interrelationship. In addition, the reaction of the host to
the bite of a disease vector may affect infectivity with pathogens and their
survival in the host.
The direct effects of ticks on animal production result from damage to
hides, tick worry, blood loss or the effect of tick toxins (Uilenberg, 1992). Direct
damage to the hide caused by the tick bite and abscesses that develop at
those sites result in appreciable loss in value of the hides. The blood loss
caused by engorging female ticks, which is responsible for a major part of this
reduction in live weight gain, may, under certain circumstances, cause severe
anemia and even death (Callow, 1978).
The indirect effects of ticks on production are through the diseases they
transmit. These diseases are distributed throughout the world on a regional
basis, depending on the distribution of the tick vector involved. Tick-borne
hemoparasitic diseases have been regarded as the most important livestock diseases in the world. These diseases have proved to be difficult to control owing to the complexity of the etiologic agents and their accompanying disease pathogenicities. All species of ticks are exclusively hematophagous in 12 all feeding stages. Several characteristics of ticks make them outstanding vectors of pathogenic agents. Their wide host ranges and tendency to feed on several hosts during their lifetime ensures ample opportunity to acquire and transmit pathogens. Their hardiness and longevity allow them to survive periods of unfavorable environmental conditions. They have a high reproductive potential, ensuring large populations and a high frequency of host-vector contact. Finally, they feed slowly and attach to the host for a relatively long period. This allows sufficient time for pathogen acquisition and transmission as well as vector dispersal by migrating hosts.
Ticks have been of considerable medical-veterinary importance
(Sonenshine, 1991 & 1994) because of their ability to harm their hosts through direct action such as tick paralysis (Kocan?) and human parasitism (Carpenter et al. 1990 & Goddard, 1989). In dogs, Rhipicephaius sanguineus (Latreille
1806) is the most host-specific and regularly found only on dogs (Garris 1991).
When it is found on other hosts such as horses, cattle, or humans, it is usually because of those hosts' close association with dogs. This tick is an efficient vector of Ehrlichia canis (Groves et al. 1975) and Babesia canis, the causative agents of canine babesiosis (Ewing 1969; Lewis et al. 1977). Canine ehrlichiosis or an Ehrlichia-\ike organism has also been found in humans, and these cases may involve transmission by tick bite (Edwards et al. 1988;
Fishbein et al. 1987; Goddard 1989; Lewis et al. 1977). Moreover, the brown dog tick also transmit Hepatozoon canis (canine hepatozoonosis); and
13 Haemobartonella canis (haemobartonellosis) and can cause tick paralysis in dogs (Arthur 1962).
1.5. Economic Importance
Of all external parasites, ticks cause the greatest economic losses in livestock production in the world today. It is well known that ticks are distributed throughout the world in all countries and different species of ticks are parasitic on many species of animals, either wild or domesticated. It is estimated that the cattle population of the world exceeds 1,500 million with about three-quarters in the tick infested or potentially tick infested habitats of the world. It is interesting and important to point out that within the context of the developing world, ticks and the diseases they transmit to livestock are a major animal health problem in almost all developing countries.
The physical damage of tick infestations is caused by several factors.
There is loss of blood amounting to 1-3ml for every tick completing its life cycle on an animal. There is irritation causing licking and scratching, rather than contended foraging. There is damage to hides. There is the predisposition of the animal to bacterial and fungal infections and other parasitism, such as screwworm attack, caused by wounds left by tick bites in the animal’s skin.
Moreover, immunosuppression facilitates the occurrence of concurrent diseases (Barriga 1999). With gross infestations, the multiplication of these factors causes a total breakdown in the physical shape of the animal.
Research in Australia have shown that an average daily infestation of 50 or
14 more engorging adult Boophilus ticks caused an annual loss in growth rate of
1.67 lb. per tick or nearly 4 tons of beef in a herd of 100 animals. An estimate of the milk loss from the presence of ticks is 40 gallons per animal or 5,300 tons of butter annually (Cattle Tick Control by Wellcome Research
Organization. 1976).
1.6. Immunity to Ticks
As in cases of attack by other pathogenic organisms, mammals are capable of exhibiting both innate and acquired immunity to arthropods.
Immunity can be developed not only to parasitic arthropods but also to predatory arthropods or arthropod fragments. Acquired immunity to tick may be roughly categories into three major groups.
1.6.1. Innate or Breed Resistance
Innate resistance is a resistance that does not depend on previous infestations and develops during the first infestation of the hosts. This resistance demonstrates various degrees of host susceptibility to tick infestations and is related to a genetic characteristic of individual or breeds.
The resistance in cattle was reported to consist of innate and acquired mechanisms (George et al. 1985). Many researchers (Hewetson 1971;
Wagland 1975, 1978) have investigated the validity of the concept of innate resistance. Breed resistance is considered to be a part of genetic resistance implicated in innate resistance (Barriga 1997). Breed resistance was considered to be an alternative to tick control programs after the first report in
15 1918 when B. microplus showed their susceptible to Zebu cattle (Bos indicus)
more than European cattle {Bos taurus). Barriga et al (1993) observed that
host’s genetic composition had a strong influence on the tick infestation and development in its life cycle. His results also indicated that natural resistance was unrelated to the ability to mount an acquired resistance. This genetic resistance has been used to control tick populations more successfully in areas with a problem of acaricide resistance and heavy tick infestations (De
Castro and Newson 1993).
1.6.2. Naturally Acquired Resistance
Naturally, acquired resistance is a resistance that becomes evident after animals had been exposed to the first natural infestation with ticks.
Acquired host resistance to ticks has been observed in the laboratory and in nature. With a model system of guinea pigs as host and any of several species of ixodid ticks, an infestation with ticks will produce virtually complete immunity to such a successive infestation in some tick species.
This has been well shown with D. vanabiiis, the American dog tick, and with A. americanum. This acquired resistance also has an essential role in regulating B. microplus numbers on Australian cattle (Riek, 1962) and in the seasonal numbers of larval and nymphal /. persuicatus on various host species and on different age groups in a single small mammal species. The percentage of engorged and detached larval /. persuicatus was much lower
16 from rodents trapped in natural habitats with numerous ticks of this specie
than from rodents trapped in places with few of these parasites.
Acquired resistance by vertebrates to tick and innate resistance are
rarely absolute. They are manifested in lower infestation rates on animals
subject to repeated ixodid attack, retarded tick development on these animals,
higher tick mortality rate, reduced engorged tick weight, and lower female egg
production (Trager, 1939a, and b; Riek, 1962; Barriga et al. 1991a). With the
guinea pig model with either Dermacentor or Ambiyomma, acquired immunity
to nymphs and adults stages is expressed more in a reduction in engorged weight of the nymphs and adult females than in a reduction in the number that
are able to attach and feed. This reduction in amount of blood taken
secondarily results in a reduction in successful molting by nymphs to the adult stage and a reduction in the number of egg laid by the adult females (Trager,
1986).
When ticks feed on rabbits or sheep after a batch of the same tick species had fed on them, the larval and nymphal weight was significant lower than during the first feeding. Longer feeding and developmental periods were also observed in all stages. Progeny of these females had low viability.
Engorged females weighed less and greater flattening changed their body proportions. In many female detaching from animals that had been used twice as hosts, the gut contents were sometimes pale yellowish-grey in contrast to the first feeding when they were usually dark-brown. When feeding for a
17 second time, the female dry weight decreased when feeding on sheep
(Balashov, 1972; Barriga et al. 1991a,b; 1993; 1995).
Tick feeding induces host immune regulatory and effector pathways
involving antibodies, complement, cytokines, antigen-presenting cells, and T-
lymphocytes. (Allen et al. 1979; Wikel 1979; Brossard et al. 1982; Whelen &
Wikel 1993; Papatheodorou & Brossard 1987). Initiation of tick feeding on a
previously un infested animal presents salivary immunogens to cells in the
epidermis and dermis at the bite site. The types of immunogens introduced
into the host change during the course of engorgement. Proteins and other
immunogenic molecules in tick saliva can be processed and presented to
antigen-specific T lymphocytes in the epidermis (Langerhans cell), dermis
(macrophages), and draining lymphnodes (macrophages, dendritic cells)
(Allen et al. 1979; Nithiuthai & Allen 1984; 1985). The immune resistance
mechanisms of tick infestations are basically to reject subsequent re-
infestation with these ectoparasites. These mechanisms are associated with acute and delay cutaneous accumulation of neutrophils and basophils (Allen
1973; Tatchell & Moorhouse 1970). Immediate and delayed type hypersensitivity reactions occurred on repeated exposure of the host to the salivary antigens injected by feeding ticks (Willadsen, 1976; Askenase 1977;
Brown et al. 1982; Ribeiro 1989). Abnormal conditions for ticks feeding on repeatedly infested hosts were associated with changes in host skin reactions at the attachment sites.
18 Salivary gland derived molecules have antihemostatic, vasodilatory,
antiinflammatory, and immunosuppressive properties (Ribeiro and Spielman
1986; Ribeiro et al 1985; Ribeiro 1987). Several tick salivary-gland factors
appear to have more than one biological function. Repeatedly fed female
Hyalomma asiaticum on experimental rabbits and found that leukocytic
infiltration, dermal edema, and rapid proliferation of connective tissue at
attachment sites disturbed normal feeding (Riek, 1962). However, the general
inflammatory response with predominating hemorrhagic features during the first feeding created a favorable situation for tick engorgement. Feeding disturbance and death of most Boophilus microplus on resistant animals are explained by specific skin reactions at the attachment site (Barriga et al. 1993;
1995). When ticks were fed on susceptible animals, there was haemorrhage in the dermis and typical tissue degeneration and cell necrosis in the immediate vicinity of the basis capituli. Slight edema was also present; leukocytic infiltration did not extend deep into the dermis and polymorphonuclear cells predominated rather than eosinophils. Intense proliferation of the Malpighian layer of epidermis and dermis often lead to encapsulation of mouthparts which completely prevent blood and lymph from entering into the tick. Cutaneous reactions caused by repeated tick feeding are considered to be host defense response after sensitization by tick salivary antigens.
The allergic reaction is one of the important sign of resistance.
Histamine, in particular, contributes to immunity acquired naturally against
19 ticks (Kemp & Bourne, 1980; Brossard et al. 1982). In resistant animals, there is a rise in blood histamine 48 hours after tick attachment (Chinery and Ayitey-
Smith 1977); it falls between the fourth and seventh day as the tick drop off. In moderately resistant cattle, histamine level does not increase very much on tick attachment. In receptive animals, there is no change in the histamine content. Tick infestations of rodents caused a local basophilic infiltration that for a time was believed to be the responsible for the early rejection of the arthropods in resistant animals (Brown et al. 1981). Cattle resistant to
Boophilus ticks and challenged with homologous larvae, however, develop skin infiltration poor in basophils but with an abundance of degranulated mast cells and eosinophils (Barriga 1995). Resistance may be spontaneous or acquired following infestations, due to the development of cutaneous hypersensitivity.
1.6.3. Induced Acquired Resistance
1.6.3.1. Crude Whole Tick Materials
There is a variety of crude extracts of tick tissue currently demonstrated to induce immune protection to tick infestations (Trager, 1939;
Brossard, 1976; Wikel, 1981; Johnston et al. 1986; Mongi et al. 1986;
Opdebeeck et al. 1988). Bagnall (1975) consistently produced immunity by subcutaneous injection of larval extract of Ixodes holocyclus in guinea pigs resulting 29-68% of rejection of larval challenge compared with unvaccinated controls. Immunization with crude antigen preparations, however, does not
20 appear to be an optimal approach to the Induction of resistance. Barriga et al
(1991) suggested that competition between irrelevant and protective antigens
might evolve in tick infestations since he observed an inverse relationship
between antibody responses and resistance. Obtaining purified antigens and
using them with different adjuvant system should significantly advance the
search for an efficient method of artificial immunization (Szabo et al. 1995).
1.6.3.2. Salivary Gland Immunogens
It appears that antigens that induce resistance reside in the salivary
glands and midgut of ticks (Wikel, S. K. 1981; Wikel et al 1987; Willadsen et
al. 1988). The sera of tick resistant rabbits contain antibodies which bind
specifically to tick salivary gland tissue (Bowessidjaou et al. 1977), and
passive transfer of such sera confers on the recipients a degree of resistance
to tick feeding (Brossard, 1977). Trager (1939), Wikel (1981), Brown et al
(1984) have shown that salivary gland extracts can artificially induce tick
resistance in guinea pigs. In view of the complexity of the Ixodid salivary
glands (Binnington, 1978; Fawcett et al. 1981), it seems likely that one of
secreted components could induce protective immune responses.
The immunogenicity of the salivary gland was demonstrated first by
Allen & Humphreys (1979) and according to Brown et al (1984), Brown &
Askenase (1986), Shapiro et al (1989), and Jaworski et al (1990), Sahibi et al
(1997) the antigens responsible for the induction of host resistance in natural infestations appear to be of salivary gland origin. Ixodid tick saliva has been
21 the subject of considerable investigation, in all known life cycles of tick-borne
pathogens, initial infection of tick tissues occurs in midgut epithelial cells and
transmission is effected as ticks feed after parasites have developed and
multiplied in salivary glands. Multiplication in salivary glands seems precisely
co-ordinated with the tick feeding cycle; infective stages develop and are
transmitted during feeding. Therefore, interruption of salivary gland function
could prevent transmission of these tick-borne pathogens to the hosts (Kocan,
1995). The immune response elicited is due to many of the proteins detected
in tick saliva or in salivary gland extract (Kaufman, 1989). Salivary gland proteins of tick changed during feeding (McSwain et al. 1982). McSwain observed that numerous changes during attachment, feeding and copulation process were the primary stimuli for the synthesis of major new kinds of salivary gland proteins. Many tick saliva proteins have been demonstrated to be immunogenic to the mammalian host (Sonenshine 1991; Barriga et al
1991.), and anti-tick saliva antibodies have proved to be biological markers of tick exposure in humans (Schwartz et al. 1990).
Several attempts have been made to artificially immunize hosts with tick extracts. Trager (1939b) was the first to obtain partial immunity in guinea pigs with an injection of D. variabilis extracts. Kohler et al. (1967) reduced the number of Hyalomma anatolicum excavatum maturing on a rabbit by prior immunization with salivary gland extract. More convincingly, Schneider et al.
(1971) found that immunization of tortoises with a homogenate of unfed
22 nymphs of Ambiyomma testudinis prevented infestation. Garin and Grabarev
(1972) reported success in immunizing with subcutaneous injections of
Rhipicephaius sanguineus saÏNary glands. Brossard (1976) subcutaneously
injected calves with partially engorged salivary glands of adult female
Boophilus micropius. These calves were infested and gave a lower yield of engorged ticks than did the control.
Purified salivary gland antigens of D. andersoni injected into rabbits stimulated chemotactic activity, mediated either directly or indirectly through the activation of C5 (Gordon & Allen, 1991). These authors proposed that depletion of C5 may reduce the formation of complexes (C5-C9) that attack membranes in the tick gut and that decomplementation of the blood meal would also decrease the protective effects of complement-fixing antibodies in the blood meal.
1.6.3.4. Midgut Antigens
Allen and Humphreys (1979) immunized guinea pigs with an extract of either midgut or reproductive tract derived from partially fed Dermacentor andersoni females. Resistance induced with midgut antigens was characterized by inhibition of egg laying, and no larvae hatched from the eggs that were laid. The effects were more dramatic in the guinea pigs immunized with reproductive tract extract since ticks failed to engorge and produce eggs.
Ackerman et al (1980) used whole tick gut from 2-3 day fed D. vanabiiis immunized rats and challenged with the same specie. This had a significant
23 level of resistance to challenge infestation. These cleared evidences have supported the possibilities of antigens derived from tick tissues other than salivary gland is capable of inducing resistance against ticks.
A series of studies involving immunization of cattle against B. microplus have been reported by using midgut antigen known as ‘concealed antigen'
(Johnston et al. 1986; Agbede and Kemp, 1986; Kemp et al. 1986; Willadsen et al. 1988; Willadsen and Kemp 1988; Willadsen and McKenna 1991;
Willadsen et al. 1991 ; Willadsen et al. 1993). Cattle were immunized with midgut antigen prepared from adult female B. microp/us. Damaged ticks obtained from immunized animals had host erythrocytes in their hemolymph indicating a link between induced resistance and tick gut damaged.
Histological examination of tick feeding on immunized animals showed extensive damage to tick gut cells, and ruptured gut caecae were observed
(Agbede and Kemp, 1986). It is cleared from this study that components of the tick gut surface could be most suitable targets for host-immune effector elements (Kemp et al. 1986).
Sahibi et al (1997) compared immunizing power of salivary or intestinal extract, and field infestation of Hyalomma marginatum marginatum in cattle.
Their results indicated that salivary extract immunization protected from tick attachment and inhibited tick feeding and fertility whereas intestinal extract immunization effected mostly fertility.
24 1.6.3.5. Passive Transferred Resistance
Host antibodies have a role in resistance to ticks and they can be
transferred to naive animals and conferred protection to challenge with these
ticks. Trager (1939) found that resistance could be transferred passively by
intraperitoneal inoculation of serum from hyperimmune animals into naïve
animals. Most of the ticks that fed on the immunized animals were abnormally
small and died. Although he was initially unable to demonstrate the presence
of antibody, complement-fixing antibodies were reported subsequently.
Passive transfer of laboratory animals resistance to ixodid ticks has been
established for guinea pigs infested with D. variabilis (Ackerman et al. 1981), /.
holocyclus (Bagnall, 1975), A. americanum and R. sanguineus (Brown and
Askenase, 1981), and for rabbits exposed to /. ricinus (Brossard, 1977) and R.
appendiculatus (Rubaire-Akiki and Mutanga, 1980). B. microplus (Roberts and
Kerr 1976)
1.6.3.8. Immunomodulation
Immunomodulation was early observed in dogs infested with I. holocyclus by Bagnall (1975) and in cattle infested with B. microplus by Reich and Zorzopulos (1980). This phenomenon also reported by many investigators. Immunosuppression was induced in guinea pigs infested with D. anc/erson/(Wikel et al. 1978; Wikel 1982; 1983; 1985). Barriga at al (1991a) observed that A. americanum ticks infested in sheeps often fared better in the subsequent infestations than in the first infestation and the anti-tick antibodies
25 decreased with the number of infestations indicating that the infestations
affected the immunological responsiveness of the host. Barriga et al (1991b)
also observed that sheep exhibiting the highest anti-tick resistance also
demonstrated the lowest antibody response. Barriga et al (1992) investigated
the immunosuppression in dogs infested with demodectic mange and found
the evidence that immunomodulation was induced by the parasites. Barriga et
al (1993) concluded that B. micropius could evade host immunity in the course
of repeated infestations. Other reports of immunosuppression induced by ticks
include depression of both cellular and humoral immune responses (Wikel,
1985; Wikel & Whelan, 1986; Fivaz, 1989; Njau et al. 1990; Dusbabek et al.
1995) and interference with cytokine production (Ramachandra & Wikel,
1992). Barriga (1999) reviewed the evidences and mechanisms of
immunomodulation in tick infestations. Suppression of Thi response (cell-
mediated immunity) or Th 2 response (humoral immunity) have been observed
in both laboratory and natural model by many investigators. However, the
related mechanisms are questionable. Antigenic competition, lymphocyte
responsiveness, host cytokines modulation, and humoral effector inhibitor in the tick salivary gland were considered to be involved in this phenomenon.
1.6.3.7.Identification of Antigens Associated with Resistance
One of the most obvious mechanisms that parasites use to reduce the immune response to protective antigens is the release by the parasite of a large number of potent antigens irrelevant to resistance but able to inhibit the
26 response to the weaker relevant antigens (Barriga 1992). The clear identification and subsequent purification of the antigens responsible for the generation of resistance is essential for the development of a candidate anti tick vaccine. Identification of tick antigens that can induce resistance to tick infestation has also been a focus of many works during the last ten years. The search for tick antigens that could be used for tick vaccines has been led by many investigators, notably Ackerman et al. 1980; Wikel, 1981; Brown et al.
1984; Brown & Askenase, 1986; Johnston et al. 1986; Shapiro et al. 1986,
1987; Gordon & Allen, 1987; Opdebeeck et al. 1988; Willadsen & Kemp, 1988;
Willadsen et al. 1988, 1989; Nyindo et al. 1989; Dhadialla et al. 1990; Barriga et al. 1991b; Essuman et al. 1991, 1992. Antisera from resistant animals made by tick-tissue immunization or infestation have to recognize the effective target antigen and should be useful for antigen identification (Shapiro et al. 1989).
The salivary gland antigens have been characterised by immunoblot analysis following sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Brown,
1985, 1988; Gill et al. 1986; Shapiro et al. 1986, 1987, 1989; Gordon & Allen,
1987). The characterization of pharmacological and immunological properties of tick saliva provides a better understanding of the interrelated elements involved in acquisition and expression of host immunity to ticks.
1.6.3.8. Immunity to R. sanguineus
Very little is known about immunity to the brown dog ticks,
Rhipicephaius sanguineus, especially the properties of their salivary glands or
27 the mechanisms of immunological protection. Garin and Grabarev (1972) reported the acquired resistance developed by laboratory animals was reflected by a decline in tick engorgement weight and egg laying by adults, and reduced egg viability. Only a few researchers have worked with R. sanguineus and report less progress compared to other species of tick. Theis
& Budwiser (1974) reported sequential histopathology of R. sanguineus at host’s attachment sites and concluded that dogs did not develop resistance against tick infestations. Theis et al (1976) reported the significant changes in flow rates of cells and total protein content of lymph, between lymph drained from sites of inflammation induced by the brown dog tick’s attachment.
Mulmule (1991) observed structural and cytochemical changes in the salivary glands of R. sanguineus during feeding. Cytochemical changes suggested that salivary gland elaborated different secretory products including the main constituents of phospholipids, proteins and carbohydrates.
(a) Humoral and Cellular Immune Responses
Tick feeding can stimulate production of antibodies to antigenic determinants associated with ixodid tissues other than salivary glands. It is possible that the same antigenic determinants were present in both gut and salivary gland. Rabbits infested with H. anatolicum excavatum and R. sanguineus developed antibodies that reacted with salivary gland and digestive tract antigens (Kohler et al. 1967). Kohler demonstrated antigens in the digestive system as well as salivary glands by using immunofluorescence
28 staining with serum from infested rabbits. Chinery & Ayitey-Smith (1977) and
Chinery (1981) showed that the salivary glands of R. sanguineus contains a
histamine blocking agent that implies an efficient mechanism for regulating the amount of histamine in the host’s tissue at any particular moment to the advantage of the tick. Perhaps small amounts are released during the first period of slow engorgement and larger amounts released during the final period of rapid engorgement when such amounts may be required to cause a more intense reaction In the host, leading to the host development of resistance.
Brown & Askenase (1981) found that immune resistance to ticks is dependent on sensitized lymphoid cells or serum complements. Immune rejection of ticks is usually associated with recruitment of basophils and eosinophils in large numbers. Brown et al (1982) ‘s further investigation exposed that intravenous transfer of immune serum from resistance hosts conferred a significant level of protection to R. sanguineus and other tick species. Ben-Yakir (1989) demonstrated that host antibodies found in the haemolymph by passing through the gut wall of the tick. The passage of antibodies may actually be caused by a slight inefficiency of the digestive tract, permitting a very small fraction of ingested proteins to leak into the hemocoel, which might clarify the role of antibodies in the host resistance to these ticks.
The concentration of IgG in the haemolymph is influenced directly by the titer of antibodies in the host blood (Ben Yakir 1986) and could cause damage to
29 the tick if directed against appropriate targets in the tick hemocoel.
Puttalakshmamma et al (1994) demonstrated the cellular response in rabbits to extracts of different stages of R sanguineus. However, he did not show the relation of cellular response and resistance of hosts against R sanguineus.
Rahman et al (1992) supported the evidence of association between the increasing immunoglobulins and the development of resistance in rabbits. His results showed the increased levels of IgM, IgG and total immunoglobulins in the sera of rabbits immunized with different antigens of R sanguineus. This was sustained by Hernandez et al (1994) ‘s ELISA results that present the importance role of humoral antibody in host resistance to ticks. Anti-salivary antibodies neutralized salivary enzyme vital for tick feeding led to a direct correlation between IgG titres and the degree of resistance.
(b) Repeated Infestation
Brown et al (1982) shown that guinea pigs were capable of developing resistance against the brown dog tick and this resistance can transferred to naïve animal and gave the protection against this tick. Contradictory to rodent model, Szabo et al (1995), Bechara et al (1995) observed that repeated infestations in dogs, which are incapable of induction of resistance. The reason of this failure might come from a histamine blocking agent that might involved in the dog's tolerance, and immunosuppression was emphazised.
They suggested that lack of resistance against parasites was a common feature of natural host-parasite relationship even after repeated infestations.
30 However, Ferreira and Bechara (1995) investigated the development of
resistant in domestic and wild dogs to R. sanguineus infestations since there was the suspiction that domestic dogs might have lost their capability to react during evolution of the host-parasite association. The results of both groups had no significance differences in developing less resistance to tick infestations. Szabo and Bechara (1995) also reported the difference of local reaction against R. sanguineus infested on dogs’ skin. Dogs, unable to display resistance, reacted with a PMN neutrophil infiltration while in the resistant guinea pigs and hamster found mononuclear or eosinophilic infiltrate. Ferreira and Silva (1998) found that saliva of R sanguineus adult tick impaired T cell proliferation and enhanced host immune modulation in mice. R. sanguineus saliva profoundly inhibited T cell response to Con-A via either an effect on the
T cell themselves or inhibition of IL-2 production. This saliva also showed an impact on impairing macrophage activation by diminishing the IFN-y and reducing production of Nitric Oxide (NO) that was essential in microbicidal activity.
Inokuma et al (1997a) reported that dogs had developed resistance to
R. sanguineus after twice repeated infestations. Inokuma et al (1997) observed the suppressive effects on the neutrophil function and the host antibody production of dogs infested with R. sanguineus. Tick infestation modulated chemotaxis response of neutrophils that will enhance the possibility of immunomodulation phenomena. Inokuma et al (1998) further investigated the suppressive effects of salivary gland extract on the blastogénie responses of peripheral blood lymphocytes from dogs infested with adult R. sanguineus.
Significant suppression of T cell was observed when tested R. sanguineus salivary gland extract with Con-A and pokeweed mitogen. The results indicated that some proteins in salivary gland extract might contribute to the suppressive effects on the blastogénie response of dog peripheral blood lymphocytes.
(c) Tick Tissue immunization
Garin and Grabarev (1972) were the first investigator who demonstrated that rabbits and guinea pigs developed resistance after immunization with salivary gland. Contrast to experimental host, Bechara et al
(1994) failed to develop resistance in dogs by immunization with crude unfed adult extracts; nevertheless, he can induce resistance in guinea pigs and hamsters. He suggested that more purified tick extracts might be necessary to induce resistance in dogs against R. sanguineus. Szabo et al (1995) used intradermal testing to detect a reaction in dogs and guinea pigs, which were immunized by unfed adult extract of R. sanguineus. The results demonstrated that dogs reacted to tick extracts with a strong immediate hypersensitivity reaction and less intense in a delayed type reaction. These reactions were lost in repeated infestations in dogs because of immunomodulation caused by salivary secretions during feeding. Guinea pigs, unlike dogs, display a less intense immediate but a strong delayed type reaction.
32 Szabo and Bechara (1997) immunized dogs and guinea pigs using gut
extract of R. sanguineus and have found that gut extract induced a more
efficient immunity than salivary gland extract. This result implied that cellular
immunity might be a major part in the resistance of dogs against this tick since
cellular mediated immunity of dogs may be diminished during infestations.
This study also showed different patterns in resistance to ticks between natural and laboratory animals.
Tripathi et al (1998) immunized rabbits with unfed larvae and nymphs materials of R. sanguineus and demonstrated significant level of protective immunity to infestation with all stages of this species. This procedure was based on the concept that adult ticks were affected by immunization with antigens purified from larval extracts, to the same extent as with midgut extracts prepared from partially fed females (Opdebeeck et al. 1989). The immunized rabbits were responded for immediate type hypersensitivity reaction when tested on intradermal tests. Immediate hypersensitivity reactions resulted in influence of vasoactive amines mainly histamine, which has been found to have a direct effect on tick attachment and feeding behaviour. The levels of resistance and degree of reactions were directly related to the increased levels of histamine in the blood circulation.
(d) Identification of Immunogen Associated with Resistance
Szabo et al (1995) reported that vaccination led to the recognition of a greater number of tick antigens by both dogs and guinea pigs than of repeated
33 infestation. Sera from naïve guinea pigs recognized some tick antigens suggesting cross reactivity between tick and unrelated antigens in the rodent.
Their results also indicated that there were many different patterns of antibody production following vaccination and infestation. Hernandez et al (1995) analysed stage-specific and shared antigens derived from R. sanguineus using sera of rabbits infested by larvae, nymphs, and adults. Anti-larva sera recognized 32, 50, and 80 kDa while anti-nymph sera detected 50 and 80 kDa.
Sera from rabbits infested with adult ticks demonstrated common protein bands specifically in 32 kDa in different stages and salivary gland antigens and identified 45 and 116 kDa in male and female salivary gland. Ferreira et al
(1996) using resistant sera of guinea pigs immunized with unfed adult extract detected 97 kDa and considered this protein as responsible for the induction of anti-R sanguineus immunity in guinea pigs.
1.7. Control of Ticks
The basis of successful tick control is the prevention of the development of the engorged female ticks, which are the final stage of the parasitic life of the tick. These engorged female ticks lay large numbers of eggs, which form the foundation for the next generation. Control of the immature stages of two-and three-host ticks is also of importance, particularly in the prevention of diseases like Ehrlichiosis, Babesiosis, and Anaplasmosis.
34 1.7.1. Ecological Control of Ticks
1.7.1.1. Pasture Spelling
The movement of animals from pastures or rotational grazing is
sometimes called “pasture spelling” (Nunez et al. 1985). Pasture spelling has
been effectively in Australia for the control of Boophilus microplus (Johnston et
al. 1968; Mahoney, 1977). Pasture should be kept free of the mammalian host,
in these case cattle, until the free living larvae of ticks has died. To a large
extent, this method will not work in some areas that have different kind of ticks
such as 3 host tick species whose preadults may have other food sources and the unfed adults may survive for 2 years or more in the pasture (Newson et
al.1984). In some countries, this method, although effective, are unprofitable
because the sources of forage can not be adequately exploited.
1.7.1.2. Habitat Modification
Clearing of edge habitats by leaf litter removal, mechanical brush removal, mowing and burning vegetation demonstrates effective means of tick control in residential areas. Removing leaf litter and underbrush also eliminates tick habitats and reduces the density of small mammal hosts, like deer mice and meadow voles. Without leaf litter, ticks are denied suitable microhabitats that provide the necessary environmental conditions for survival, such as high relative humidity. Mowing lawns and other grassy areas to less than 6 inches greatly reduces the potential for human-tick contact. Not only does burning
35 kills all active stages, but burning reduces search success by destroying the
vegetation that is normally used to contact passing animals or humans as well.
1.7.2. Chemical Control of Ticks
Tick control is almost totally dependent upon use of acaricides. Several
factors are associated with successful acaricidal tick control, including type of
acaricide, ambient temperature, dosage, penetrability of canopy, extent of
coverage, susceptibility of the target tick species, tick life stage and
physiological condition. Two methods of tick control by acaricides are possible: first by application of chemicals to the surface of the animal and second by a
systemic attack of the tick by injection of the pesticide into the host and
appearance in its blood meal. Application of acaricides to the surface of
livestock through dips and spray has been the mainstay for the control of tick
infestation and the prevention of disease transmission worldwide. However, cost must be a primary consideration for the control measures in most part of the world. The acaricides used in this application are increasingly expensive.
Not only the high cost of acaricides, but also the high-accumulated acaricides deposited in the environment result is a severe pollution problem. The most commonly used pesticides for tick control are diazinon, carbaryl and chiorpyrifos. Most organophosphates and carbamates are less effective at temperatures below 70°F. Diazinon is also recommended for use indoors and outdoors to control the brown dog tick, R. sanguineus. Since acaricides have been used extensively, Rhipicephaius and Amblyomma species have
36 developed resistance to most of these acaricides (Keating, 1983; Chema,
1984).
An ever-present danger Is the development of tick populations with
resistance to acaricides. Such resistance Is well documented both In Africa
and elsewhere (Nolan et al. 1983). Development of resistance to acaricides Is
usually seen first In the one host tick, Boophilus spp. followed eventually by
two and three host ticks. 8. microplus Is proving the (most prolific producer of
resistant?). Varying degrees of resistance have been reported for all the major
chemical groups used for tick control (Solomon 1983). Matthewson (1984)
pointed out that the number of acaricides Is limited at present, development
costs are very high, and If resistance develops rapidly, companies can not
recover their high development costs. This has led many Investigators to
suggest other approaches to tick control (Sutherst 1983). In many areas of the
world Boophilus species are not the main problem but increasing of tick-borne
disease’s Incidence by the other kind of tick Is the major consideration.
1.7.3. Biological Control of Ticks
In other areas, the uses of predators or pathogens have found little success. A range of Insect predators of ticks have been Identified but not put into use (Matthewson, 1984). Birds, such as the red bill and bill oxpecker, feeding on ticks, can cause damage to cattle as well (Stutterhelm &
Stutterheim, 1980). Probably the most exciting area of biological control of ticks Is the use of the resistant cattle to control tick population (Cunningham,
37 1981). Cattle develop an acquired immunity to infestation which varies
between individuals and breeds (Seifert, 1984). Two practical applications of
host resistance to tick infestation is to select cattle with the ability to produce a
high degree of tick resistance or artificially induce tick resistance.
1.7.4. The Development of Vaccine against Tick
Control strategies used to date have had limited success. Vectors have
developed resistance to a number of insecticides and parasites have
developed resistance to drugs that were once extremely effective. These facts
underscore the need to devise novel strategies for disease control. Artificial
immunization against tick infestation may be the most attractive alternative
application to control ticks (reviewed by Kay & Kemp 1994; Barriga et al.
1994).
It has been a problem to target the stages of the parasite for vaccine development, because over millions of generations of co-evolution, the parasite has evolved ways to circumvent its host’s defence mechanisms.
Actually, arthropod vectors have only limited contact with the mammals on which they feed since, except for the mouthparts and saliva, no direct contact is made between the different organs of the arthropod vector and its host.
Arthropod have become successful disease vectors due to their ingesting the blood of their hosts and providing an environment that is permissive for survival and development of disease organisms. However, antibodies against arthropod organs in the blood meal have been proved that can be used either
38 to disrupt the biology of the vector (Agbede & Kemp 1986; Kemp et a! 1986;
Nogge & Giannetti, 1980; Willadsen et al. 1989), or to prevent these
arthropods from transmitting diseases (Billingsley 1994; Wikel 1980). Thus,
anti-tick vaccine might have a speculation in two different ways as indicated by
the following observations and demonstrated that immunization to ticks could
protect from tick-transmitted diseases of animals and humans, such as
Theileria pan/a, the cause of East Coast fever, transmitted by three-host ticks
such as Rhipicephaius spp. Animals immunized against the vector tick such
as D. andersoni were protected from tularemia, a bacterial disease transmitted
by ticks (Wikel 1980). In addition, vaccination of cattle against the cattle tick,
B. mioroplus, a one-host tick and the vector of babesiosis, theileriosis, anaplasmosis, might protect them both against these diseases and against the considerable direct damage that results from heavy infestation with ticks. This would be sufficient to take the vector from the status of epidemiological transmitter to that of only pest (Billingsley, 1994).
Trager (1939) was the first to show that resistance to a blood-sucking arthropod can be induced by immunization with arthropod tissue. Trager reported that laboratory animals acquired resistance to the blood feeding activities of ixodid ticks. Guinea pigs and rabbits are subjected to repeated experimental infestations with D. vanabilis acquired resistance which was manifested by lower weights of fed ticks, reduced feeding rates, reduced abilities of fed larvae or nymphs to molt to the next stage or reduced progeny
39 of fed female ticks. Trager also suggested that this acquired resistance was
immunologically mediated, and provided some evidence of passive transfer of resistance, artificial induction of resistance with extracts of larvae, salivary glands or digestive tracts and a degree of cross-resistance between
D. vanabilis and D. andersoni. Since Trager's original work, acquired resistance to tick infestations has been investigated in laboratory animals such as rabbits and guinea-pigs, and livestock such as sheep and bovines, as well as pet animals such as dogs by a large number of ixodid species (Brown,
1988; Wikel 1983; Wikel & Whelen, 1986; Willadsen, 1980; Allen, 1989;
Brossard et al. 1991; Barriga et al. 1991; 1993). This immunological phenomenon was expressed by a significant decrease in tick-feeding habits, and fecundity (Brown, 1985; Wikel, 1982; Wikel & Whelen, 1986; Schorderet &
Brossard, 1993; Sahibi et al. 1997).
Recent works indicated that it is feasible to attack arthropods through the use of either natural antigens (salivary gland origin) or novel antigens
(internal tissue origin). Novel or concealed antigens are not exposed to the immune system of the host during feeding. This concept has lost to support compared to the use of conservative antigens. However, important functional molecules that are exposed to the immune system of the host are likely to have lesser potency as immunogens and hence will be poor candidates for putative vaccines.
40 Allen and Humphreys (1979) had suggested previously that antigen
extracts should be generated from fed parasites because extracts from unfed
ticks failed as vaccines; they had achieved protection of the host only if the
antigens were extracted from D. andersoni female ticks that had fed an
homologous host for 5 days. Moreover, marked changes in the morphology
and physiology of tick salivary glands (Binnington, 1978), and tick gut (Agbede
and Kemp, 1985) occur during feeding. In addition, changes in the antigenic
profiles of salivary glands of ticks in response to feeding have been reported
(McSwain et al. 1982).
Brossard (1976) found that serum y-globulin concentration was significantly increased following tick infestations. Truly, specific antibody appeared following initial infestation of host with ticks, reached high titres, which were maintained throughout infestation, and then declined over a period of time once infestation was finished. However, the results of Barriga et al.
(1991) have shown that antibody responses are inversely related to the number of infestations. This pointed out the immunosuppressive effect happened during the infestation and concealed the acquired immunity.
Brossard (1977) and Bowessidjaou (1977) were working with /. ricinus and concluded that the antibody titre against ticks might not reflect the level of resistance. Likewise, Willadsen et al. (1978) measured antibody to a purified tick antigen by indirect haemagglutination; the antibody was specific, in that it was absent from unexposed animals, but its concentration did not correlate
41 with the degree of immunity of an animal and, actually, higher titres tended to
be found in host of low immunity. Tracey-Patte (1979) reported that activity of
an enzyme from B. microplus, which is secreted into the host's skin within one
hour of attachment, could be removed by a host previously exposed to the
tick. In unexposed hosts, removal does not occur. This reaction could be
antibody-dependent.
In addition to the decrease in disease transmission caused by reduced
vector numbers, the acquired resistance reaction can directly interfere with
pathogenic organism transmission by ticks (Bell 1945; Bell et al. 1979; Francis
& Little, 1964; Wikel 1980; Wikel & Allen, 1982). Wikel et al (1997) recently
infested mice with I. scapularis nymphs and observed host resistance to
transmission of Borrelia burgdorferi. Although these mice showed no signs of
resistance, they developed an hostile environment for the pathogen and
decreased the percentage of transmission. Immunology may provide
alternative methods to control ticks and tick-related disease. It might be
possible to attack ticks by immunising potential hosts with tick antigens; then
tick would imbibe blood containing antibodies directed against themselves, or
damage the tick important organs, could reduce tick viability by interfering with
feeding. The effects on ticks feeding on cattle vaccinated with crude antigenic
extracts were studied histologically (Agbede and Kemp, 1986). They found that digest cells in the gut were lysis followed by the leakage of host cells into the hemocoel. Infiltration of host leukocytes into reproductive tissue and
42 muscle led to pathological tissue damages. Interestingly, no destruction of salivary tissue was found. Such effects have never been recorded in
B. microplus feeding on cattle with strong immunity acquired from tick infestation (Roberts, 1968; Roberts, 1971). Significant damage to ticks was produced by feeding on vaccination sera, but not on sera from animals with a strong acquired immunity to ticks. Such observations clearly show that vaccination-induced and naturally acquired immunity were different phenomena in this tick-host system.
There are two important considerations before making an anti-tick vaccine. Vaccination with midgut tissue induced an obvious resistance mainly as a great reduction of tick fecundity that had the most advantage on tick population control. However, this vaccine might have a less impact on tick- borne diseases. The salivary gland vaccine is more effective in preventing feeding and partially effective in depresses fecundity, as well as interference transmitting diseases.
43 CHAPTER 2
FEEDING AND FECUNDITY PERFORMANCE OF RHIPICEPHALUS
SANGUINEUS (ACARI: IXODIDAE) FED ON DOGS AFTER MULTIPLE
INFESTATIONS AND IMMUNIZATION WITH TICK SALIVARY GLANDS OR
MIDGUT TISSUES
INTRODUCTION
The phenomenon of acquired immune resistance to ticks following infestation has been studied since the initiatory work of Trager (1939), and different inflammatory and immunological phenomena have been described for animals infested with ticks (reviewed by Brown 1988, Rechav 1992, Wikel
1996). It has been reported that plasma from cattle highly resistant to
Boophilus microplus conferred some resistance to unexposed calves (Roberts and Kerr 1976), and that infestation of guinea pigs with R. sanguineus invoked resistance to subsequent infestation that could be passively transferred to unexposed guinea pigs via immune sera (Brown and Askenase
1981). Immunization of guinea pigs with R. sanguineus salivary gland or 44 midgut tissues was also found to confer resistance to tick infestation (Garin and Grabarev 1972, Wikel 1977, Allen and Humphreys 1979).
Hosts that become resistant after multiple infestations often display immune responses to substances found in tick saliva (Willadsen 1980; Wikel
1982 a, b, 1984; Wikel and Whelen 1986). Thus, it has been proposed that immunization with salivary gland extract could induce resistance to infestation that resembles the immune protection observed after repeated infestations.
Tick feeding can also stimulate production of antibodies to ixodid tissues other than salivary glands. For example, rabbits infested with Hyalomma anatolicum excavatum and R. sanguineus developed antibodies that reacted with both salivary gland and midgut antigens (Kohler et al. 1967), indicating that tick saliva contains antigenic determinants that were present in both the midgut and salivary glands or that the host is exposed to tick products other than saliva (Brown 1988).
While salivary gland antigens may induce host resistance similar to that induced by tick feeding, another approach has involved "novel" or "concealed" antigens that are unlikely to reach the host under natural feeding conditions
(Willadsen 1987). These antigens, which are associated with the tick midgut, have been shown to reduce tick fecundity (Johnston et al. 1986, Kemp et al.
1986, Wikel 1988, Willadsen and Kemp 1988, Sahibi et al. 1997).
Characterization of these concealed tick tissue antigens resulted in development of a defined vaccine against B. microplus (Willadsen et al. 1995).
45 Some studies have shown that dogs have the ability to develop
resistance against R. sanguineus (Pogoielyi 1966, Inokuma et al. 1997), while
others have indicated that dogs are unable to acquire resistance against this
species (Chabaud, 1950; Theis and Franti 1971; Randolph 1979; Fielden et al.
1992; Bechara et al. 1994; Ferreira et al. 1995; Szabo et al. 1995). Host
resistance to infestation can be determined by measuring tick feeding and
fecundity performance parameters, and several of these parameters have
been shown to be affected by hosts that were resistant to other tick species
(Bowessidjaou et al. 1977, Brossard et al. 1982, Barriga et al. 1991, 1995,
Sahibi et al. 1997). The purpose of this project was to determine if resistance
against R. sanguineus could be induced in dogs by measuring the feeding and
fecundity performance parameters of R. sanguineus females fed on dogs that
were exposed to repeated infestations or immunization with midgut or salivary
gland extracts.
MATERIALS AND METHODS
Ticks. Rhipicephaius sanguineus adults were obtained from the Oklahoma
State University Medical Entomology Laboratory, and maintained at 25 °C with a 12 h photoperiod and 90% relative humidity. Individual engorged females were kept individually in glass tubes throughout oviposition, and eggs were stored separately until hatching.
46 Antigen Preparation.Tissues were collected from 25 adult ticks (15 females and 10 males) for each immunization. Tick salivary gland (TSG) antigen was prepared from male and female R. sanguineus that had fed for either 3 or 5 d on a guinea pig. Ticks were placed into 0.15 M phosphate buffered saline, pH
7.4 (PBS) and opened along their dorsal surface. Salivary glands were removed, dissected free of other tissues, placed into PBS at 4 °C and then suspended in 1% SDS and 5% 2-mercaptoethanol prior to incubation in a water bath at 56 °C overnight and boiling for 5 minutes. The solution was cooled to room temperature, transferred into a 12,000-14,000 molecular weight cut off dialysis tube (Spectra/Por7, Denver, CO), immersed into 1 liter
PBS and left at 4 °C on magnetic stirrer overnight (PBS was changed every 4-
6 h). This mixture (0.5 ml) was filtered and mixed with 0.5 ml of complete or incomplete Freund's adjuvant H37Ra (Difco laboratories, Detroit, Ml) immediately prior to immunization of the hosts.
Tick midgut samples (TMG) were also removed at 4 °C in PBS. These organs were disrupted for 30 s in PBS at 4 °C with a tissue homogenizer followed sonication for 15 s (Model 300 Sonic Dismembrator, Fisher Scientific,
Pittsburgh, PA), set at 35% and 60% output power, a total often times. The homogenates were dialyzed in PBS at 4 °C overnight and centrifuged at
16,000 G for 30 min at 4 °C. TMG preparations were sterilized with a 0.45 ^m filter (Millipore, Bedford, MA) and 0.5 ml mixed thoroughly by sonication with
47 0.5 ml of complete or Incomplete Freund's adjuvant at a final prior to
immunization.
Experimental Hosts. Nine Beagle dogs were purchased from Harlan
(Indianapolis, IN) for this experiment. All dogs were 8 month old females that weighed 6-8 kg, and were randomly assigned to three treatment groups.
Immunization of Dogs. The two groups of immunized dogs were inoculated three times at 21 d intervals intradermally with the TSG or TMG preparations.
Each dog received TSG or TMG collected from 25 ticks per immunization, which was mixed with Freund's complete adjuvant for the initial immunization or incomplete adjuvant for the second and third immunizations. The third group was infested five times at 21-d intervals.
Challenge Infestation.Dogs immunized with TSG or TMG were challenged by tick infestation seven days after the third immunization and a second time
21 d later. All infestations consisted of 80 female and 40 male unfed adult R. sanguineus per dog. Ticks were placed inside a feeding bag made from an orthopedic stockinet that was adhered with tissue glue (Ellman glue, Hewlett,
NY) to the shaved scapular regions of the hosts. Elizabethan neck collars and body suits were placed on all animals to prevent grooming. In order to avoid the escape of ticks during experiments, hosts were kept in cages placed in an individual cell surrounded by petroleum jelly. Daily observations were performed to measure the tick performance parameters that are described below.
48 Tick Performance Parameters.Ticks were examined daily and detached,
engorged females were removed and individually maintained in a tick chamber
until oviposition was completed. Egg clusters were weighed and maintained
until hatching was achieved. The following biological parameters related to
female tick feeding and reproductive performances were determined during
each infestation; feeding period, engorged female weight, pre-oviposition
period, oviposition period, egg mass weight, and egg incubation period. These
parameters were used to calculate feeding and fertility indices as previously described (Barriga et al. 1991, 1995). Engorged female weight was measured immediately after detachment. Egg masses were weighed 21 d after tick detachment. The engorgement period was assumed to be the time that elapsed from the liberation of ticks onto the host until their detachment at partial or full engorgement. The pre-oviposition period was the time that elapsed from the detachment of the female tick until the beginning of oviposition. The oviposition period was the time that ticks started laying eggs until they finished. The egg-incubation period was the time from the beginning of oviposition until the beginning of hatching of larvae. The feeding efficiency index was calculated by the dividing the weight of each engorged female by her feeding period, and the fecundity efficiency index was calculated by dividing the weight of each egg mass by the weight of the respective female.
Statistical Analysis.Data from biological parameters were compared between treatment groups using multivariable analysis of variance procedures.
49 All analyses assessed the effect of treatment group adjusted for the effect of multiple infestations. Pairwise comparison of LS Means was accomplished with the Tukey-Kramer method. We consider as significant those differences with P < 0.05.
RESULTS
Performance ofR. sanguineus after multiple infestations.Repeated infestation alone reduced all of the tick feeding performance parameters, with host resistance was initially expressed after the third infestation (Fig. 2.1). The number of engorged females dropped significantly from a mean of 54.33 ticks/dog (infestation 1) to a low of 26.33 ticks/dog (infestation 4; P<0.014), followed by an increase back to 52.67 ticks/dog at infestation 5. Feeding periods increased from 7.82 d (infestation 1) to 8.51 d (infestation 3; P<.
0283), 8.61 d (infestation 4; P<. 0042) and 9.01 d (infestation 5; P<. 0001).
Engorgement weights fell from 230.12 g (infestation 1) to as low as 189.83 g
(Infestation 4; P<0.0001). Feeding efficiency indices dropped from 28.82
(infestation 1) to 24.53 (infestation 3; P<. 0144), 24.09 (infestation 4;
P<0.0001) and 21.75 (infestation 5; P<0.0001).
Repeated infestation did not significantly affect several fecundity parameters including tick mortality, failure to oviposit, and egg viability
(Fig.2.2). However, the pre-oviposition (4.81 d), oviposition (14.53 d), and egg incubation (20.70 d) periods were lengthened from infestation 1 to as much as
5.39 d (infestation 5; P<0.0002), 18.16 d (infestation 5; P<0.0001) and 22.53 d
50 (infestation 5; P<0.0001), respectively for this group. Egg mass weight
dropped from 136.52 mg (infestation 1) to 106.59 mg (infestation 4;
P<0.0021).
Comparative performance after feeding on immunized or
repeatedly infested dogs.Immunization with TSG or TMG impacted several feeding performance parameters (Fig. 2. 3). The mean number of engorged females in the repeated infestation group was 51.20 ticks/infestation, which was higher than those that engorged on dogs immunized with TMG (33.67 ticks/infestation; P < 0.001) or TSG (32.83 ticks/infestation; P < 0.0006). The mean feeding period of the repeated infestation group (8.42 d) was only greater than that of the TSG-immunized group (9.55 d, P < 0.0001). The weights of engorged females throughout the course of the repeated infestations was 204.06 mg/tick, which was greater than the average of all those fed on dogs immunized with TSG (174.66 mg/tick; P < 0.05), but there was no significant difference between the engorged weight of females from repeatedly infested dogs and those from TMG-immunized dogs.
Host immunization with TMG or TSG also impacted several
R. sanguineus fecundity parameters compared to repeated infestation alone
(Fig. 2. 4). The pre-oviposition, oviposition and egg incubation periods all were
5.05 d, 16.61 d and 21.37 d, respectively, for the repeated infestation group,
5.25 d, 18.67 d (P < 0.0001) and 19.33 d (P < 0.0001), respectively, for the
TMG-immunized group and 5.2 d, 18.2 d (P < 0.0001) and 19.31 days (P <
51 0.0001), respectively, for the TSG-immunized group. The mean egg mass
weight produced during repeated infestations was 114.53 mg/ticks, which was
greater (P < 0.0001) than those fed on TMG-immunized dogs (86.46 mg/tick).
Performance during different challenge infestations.Further
comparison of tick performance during the first and second challenge
infestations provided insight to potential mechanisms and sustainability of any
resistance induced by immunization with tick tissues. For the feeding
performance parameters (Fig. 2. 5), female ticks reaching engorgement dropped from a mean of 54.33 ticks/dog, after infestation of previously
unexposed dogs, to 28.33 ticks/dog (challenge infestation 1; P< 0.0256) and
39.00 ticks/dog (challenge infestation 2) after feeding on dogs immunized with
TMG. Engorged female numbers dropped to 39.00 ticks/dog (challenge infestation 1) and 26.67 ticks/dog (challenge infestation 2; P< 0.0155) after feeding on TSG-immunized dogs. Feeding periods increased from 7.82 d to
8.45 d (challenge infestation 1; P<0.0215) and 8.82 d (challenge infestation 2;
P<0.0001) for ticks fed on TMG-immunized dogs (Fig. 5B), and to 9.08 d
(challenge infestation 1; P<0.0001) and 10.02 d (challenge infestation 2;
P<0.0001) for ticks fed on TSG-immunized dogs. Female R. sanguineus fed on control dogs had a mean engorgement weight of 230.12 mg, which dropped significantly to 148.19 mg (P<0.0001) and 201.13 mg (P<0.0488) for challenge infestations 1 and 2, respectively, when ticks fed on TSG- immunized dogs. Engorgement weights were reduced only slightly to 215.45
52 mg and 213.31 mg for ticks fed on dogs immunized with TMG. The decreased engorgement weights of female ticks fed on TSG-immunized dogs resulted in the lowest feeding efficiency index among the three groups.
Some fecundity performance parameters also varied between challenge infestations (Fig. 2. 6). Pre-oviposition periods were lengthened from 4.81 d for ticks fed on naïve dogs to 6.00 d (P<0.0001) and 6.08 d
(P<0.0001) after the second challenge infestation to immunization with either
TMG or TSG, respectively. The 14.53 d oviposition period of the control group increased to 17.84 d (challenge infestation 1; P<0.0001) and 19.5 d (challenge infestation 2; P<0.0001) for females fed on TMG-immunized dogs, and to
18.63 d (challenge infestation 1; P<0.0001) and 17.78 d (challenge infestation
2; P<0.0001) for females fed on TSG-immunized dogs. The egg incubation period prior to hatching was reduced from 20.70 d for the controls to 17.88 d
(P<0.0001) and 17.63 d (P<0.0001) for ticks fed on TMG- and TSG- immunized dogs, respectively after the first challenge infestation, but this parameter was not significantly different for either group after the second challenge infestation. The egg mass weights produced by control ticks averaged 136.52 mg, which dropped to 70.58 mg (challenge infestation 1 ;
P<0.0001) and 102.34 mg (challenge infestation 2; P<0.0023) produced by ticks fed on TMG-immunized dogs. However, egg mass weights did not drop significantly for ticks fed on dogs immunized with TSG (113.51 mg after challenge infestation 1 and 113.11 mg after challenge infestation 2).
53 R. sanguineus exposed to TMG-immunized dogs deposited fewer eggs (P <
0.0001) than those in the control group. TMG and TSG-immunized group affected fecundity efficiency indices (P<0.05) demonstrated both antigen preparations reduced tick fecundity through different means (Fig. 2. 4 H and
Fig. 2. 6 H). No significant differences were determined among the three groups for mortality, failure to oviposit, and egg viability.
DISCUSSION
The emergence of tick resistance to acaricides, the accumulation of these chemicals in the environment and their rising costs have resulted in searching into alternative methods of control based on the use of host defense mechanisms (Utech et al. 1978). The purpose of this study was to investigate the potential for inducing resistance to R. sanguineus in the canine host.
Several previous investigations have indicated potential applications for inducing protective immunity against ticks. Wikel et al. (1997) demonstrated that infestation with Ixodes scapularis resulted in protection against subsequent transmission of Borrelia burgdorferi from infected ticks, and immunization against Dermacentor andersoni protected rabbits from infection with Francisella tularensis (Bell et al. 1979). A vaccine against Boophilus microplus, a highly adapted one-host tick of cattle, has been developed with the midgut associated immunogen Bm 86, and has been shown to reduce the engorgement weight and egg laying capacity of engorged female ticks
(Willadsen et al. 1995). The success of the B. microplus vaccine indicates that
54 it may be possible to induce immune protection against R. sanguineus in dogs
as it has been done with guinea pigs (Bechara et al. 1994, Szabo & Bechara
1997). Immune resistance to R. sanguineus may reduce the fecundity and
subsequent burdens of the ectoparasite, resulting in less direct damage due to
heavy infestation of the host and perhaps curtailing the transmission of tick-
borne pathogens.
Mammals develop an immunological response to tick secretory
products during infestation (Wikel and Bergman 1997). The sera of tick-
resistant hosts have been shown to contain antibodies that bind specifically to
tick salivary gland and midgut tissues (Ackerman et al. 1981), and passive
transfer of immune sera and peritoneal exudate cells from tick-resistant hosts
conferred resistance to tick feeding (Brossard and Girardin 1979; Brown and
Askenase 1981;Askenase et al. 1982). Specific antigens from tick salivary
glands and midgut have been associated with protective immunity against
different tick species, and these tissues have been used to induce resistance to ticks in guinea pigs (Garin and Grabarev 1972; Wikel 1977; Allen and
Humphreys 1979; Nyindo et al. 1989). Better understanding of the immune
mechanisms underlying host resistance to these parasites, and identification
of the antigens targeted by these mechanisms, could lead to an effective approach to immunoprophylactic control of ticks.
In addition to identification of protective immunogens, tick mechanisms of manipulating the host immune system must also be considered (Wikel
1982a,b and 1988). These immunomodulation mechanisms seem to be the 55 most efficient under natural conditions, when hosts seemingly possess little
measurable resistance against tick infestations (de Castro and Newson 1993;
Randolph 1979; Fielden et al. 1992). This phenomenon may explain why
reduced tick performance parameters in this investigation were often
recovered in subsequent infestations. For example, after the first challenge
infestation, the number of engorged females recovered from dogs immunized
with TMG was significantly lower than those from naïve controls or dogs
immunized with TSG. However, after the second challenge infestation, the
number of engorged females recovered from dogs immunized with TMG was
not significantly different from those from naïve controls. Similar performance
recoveries were observed for the engorged weight of females fed on TSG-
immunized dogs, the egg mass weight from females fed on TMG-immunized
dogs and for the number of engorged ticks recovered after repeated
infestations. Conversely, fewer engorged females were recovered from dogs
immunized with TSG after the second challenge infestation, possibly due to a
booster effect of the salivary immunogen(s) responsible for reducing this
parameter during the first challenge infestation.
The reduction in both feeding and fecundity performances after the third
repeated infestation in this investigation indicated that a trend of protective immunity was developing in dogs without immunization with tick tissues.
Development of resistance due to successive infestations appeared to be less pronounced than direct immunization with TSG or TMG, but this is not surprising since the dog is the natural host to which R. sanguineus is highly 56 adapted. The resistance manifested by repeated infestation affected both
feeding and fecundity performances in a manner similar to that of the TSG
immunization group. This also is not surprising since the majority of antigens
released into the host during feeding are probably secretory products of the
tick salivary glands.
TMG-immunized dogs developed resistance that affected the tick engorgement number, feeding period and several fecundity parameters.
Some of these ticks were only partially attached and not feeding or detached when only partially engorged, resulting in a decreased number of engorged females and increased mortality. Those ticks that did engorge appeared to have a lower conversion rate of blood meal to egg mass since immunization with TMG had no effect on engorged weight, but did reduce egg mass weight, as indicated by the fecundity efficiency indices of these ticks. The canine immune response to TMG may have been cross-reactive to other tick vital organs such as the reproductive system, indicating that the R. sanguineus midgut antigen preparation contained antigens important for induction of immunity targeted toward reducing tick fecundity.
Others have shown that tick midgut is permeable to host immunoglobulins, and that these antibodies enter the tick hemolymph and can bind to antigens associated with other internal organs of the tick (Ben-Yakir et al. 1985; Wang and Nuttall 1994). Additionally, leakage of the damaged midgut could have exposed host reactive elements to the ovary, uterus and salivary glands as reported by Agbede and Kemp (1986). These host immune 57 effectors that are present in the bloodmeal could bind to antigens associated with the reproductive organs, syngangiion, and muscle tissue and could have a detrimental effect on the tick.
Immunization with TSG impacted both feeding and fecundity performance parameters. Ticks fed on these dogs had a reduced engorgement weight that resulted in lower feeding efficiency and higher fecundity efficiency indices. Interestingly, while the female engorgement weight was reduced for ticks fed on TSG-immunized dogs, the egg mass weights were not reduced, thus the fecundity efficiency index was significantly higher than the other groups. Conversely, females fed on TMG-immunized dogs did have a lowered fecundity efficiency index despite engorgement weights that were not significantly different from the naive controls. These results indicated that immunization with either TMG or TSG influenced tick fecundity, but through different mechanisms: immunization with TSG apparently reduced bloodmeal uptake but did not affect egg mass weights, while immunization with TMG did reduce egg mass weights perhaps by interfering with bloodmeal conversion into egg mass.
This study was the first to our knowledge to directly compare the performance of R. sanguineus females fed on dogs exposed to repeated infestation to immunization with TSG or TMG. It was found that ticks fed on dogs immunized with TSG or TMG had greater reduction in feeding and fecundity than ticks fed on dogs that were exposed to repeated infestations.
These results indicated that these dogs developed immune resistance to 58 R. sanguineus. Although the resistance observed did not result in complete mortality or reduction in fecundity, it was indicative of the potential to establish protective resistance in dogs.
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65 Number Engorged Engorged Weight 80 -I 250 n 230 . O) £ 2 1 0. ■g 40 - 190 -
20 170 1 2 34 5 Infestation Number Infestation Number
B Feeding Period Feeding Efficiency Index
9 .5 -I
(A 30 . (0>« a 25 -
7.5 20 1 2 3 4 5 1 2 34 5 Infestation Number Infestation Number
Figure 2.1: Feeding performance parameters of R sangu/net/s females during repeated infestations on dogs. Dogs were infested five times by 80 female and 40 male ticks per infestation at 21 d intervals. After engorgement, the detached females were immediately weighed and incubated separately to observe feeding performance parameters of each individual including the engorged number (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Results are shown as Least Square (LS) Means ± Standard Error; different letters in each panel indicate significantly different LS Means (P < 0.05).
66 Failure to OvipositMortality
2 3 4 2 3 4 Infestation Number Infestation Number
Pre-oviposition Period Oviposition Period 20
» SÆ • 18 . Q 16
14
1 2 4 53 2 3 4 Infestation Number Infestation Number
Non-Viable Eggs Egg Incubation Period 20 23
22 • 12
“ 21 - 8
4 20 12 3 4 5 1 2 34 5 Infestation Number Infestation Number G Egg Mass Weight H Fecundity Efficiency Index 160
140 2.5 ■ 1 120 100
80 2 3 4 1 2 3 4 5 Infestation Number Infestation Number
Figure 2.2: Fecundity parameters of R. sanguineus females during repeated infestations on dogs. Dogs were infested, ticks collected and results are reported as described for Fig. 1. Fecundity parameters measured included the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre-oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by the weight of the egg mass produced by that same tick.
67 B Number Engorged Feeding Period 70 10 b
0 a 9.5 -f- ) 1 50 - esr 9 a a 1 a b b O 8.5 4 -f-
30 8 Repeated Midgut Salivary Repeated Midgut Salivary Infestation Gland Infestation Gland
Engorged Weight Feeding Efficiency Index 230 30 a 210 a 26 - -t- aiI E 22 - b 190 b 18
170 14 Repeated Midgut Salivary Repeated Midgut Salivary Infestation Gland Infestation Gland
Figure 2.3: Feeding parameters of R. sanguineus females from all infestations fed on immunized or repeatedly infested dogs. In the immunized groups, dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The immunized dogs were subjected to two challenge infestations with 80 female and 40 male ticks per infestation, separated by a 21 d interval, one week after the final immunization. Dogs in the repeated infestation group were infested five times at 21 d intervals with 80 female and 40 male ticks per infestation. The bars in each panel represent the Least Square (LS) Mean ± Standard error of tick performace parameters from all infestations in the repeated infestation, midgut, and salivary gland immunized groups. Feeding performance parameters of each individual included the number engorged (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Different superscript letters represent significantly different (p < 0.05) measurements within each panel.
68 Mortality Failure to Oviposit
E S I ' z Z 6 ■5 3 I » ED. 1 m Repeated M idgut Salivaiy Repeated Midgut Salivary [nfestation Giand Infestation Gland
Pre-oviposftion Period Oviposition Period 6.4 b 20 b 6.3 19 4- O« 18 6.1 17
6 r f i 16 Repeated Midgut Salivary Repeated Midgut Salivary Infestation Gland Infestation Gland
Non-Vfable Eggs F Egg incubation Period 12 23 à. 11 10 I? C s : 4 18.6 Repeated Midgut Salivary Repeated Midgut Salivary infestation Gland Infestation Gland
Egg mass Weight Fecundity Efficiency Index 130 a * 0.9 • 110 - 0.7 • b Do 0.6 - 0.3 ■ 70 — 1 0.1 • n Repeated Midgut Salivary Repeated Midgut Salivary infestation Gland Infestation Gland
Figure 2.4: Fecundity parameters of R. sanguineus females from all infestations fed on dogs that were repeatedly infested or immunized with tick tissues. Feeding parameters of R. sanguineus females fed on immunized or repeatedly infested dogs. Dogs were repeatedly infested five times or immunized three times with tick salivary glands or midgut followed by two challenge infestations. The bars in each panel represent the Least Square (LS) Mean ± Standard error of ticks from all infestations in the repeated infestation, TMG, and TSG groups. Fecundity performances measured included the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre-oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by weight of the egg mass- produced by that same female. Different superscript letters represent significantly different (p < 0.05) measurements within each panel.
69 Number Engorged B Feeding Period 70 1 11
10 H (/> a 8
Repeated Midgu Salivary Repeated Midgut Salivary Infestation Gland Infestation Gland Engorged Weight D Feeding Efficiency Index 270 -, 35 1 240 -
210 -
180 -
150 -
Repeated Midgu Salivary Repeated Midgu Salivary Infestation Gland Infestation Gland
Figure 2.5: Feeding parameters of R. sanguineus females from individual infestations of dogs immunized with tick tissues. Dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The dogs were subjected to two challenge infestations with 80 female and 40 male ticks per infestation, separated by a 21 d interval, one week after the final immunization. Values from the first two repeated infestations are included as controls. Engorged, detached females were immediately weighed and incubated separately to observe feeding performance parameters of each tick, parameters included the number engorged (A), feeding period (B) and weight at engorgement (C). The feeding efficiency index (D) was calculated by dividing the body weight of each engorged female by the feeding period for that same female. Closed and open bars represent the first and second challenge infestations, respectively. Results are shown as Least Square (LS) Means ± Standard Error; different superscript letters in each panel indicate significantly different LS Means (p < 0.05).
70 Mortality Failure to Oviposit
Repeated Midmit Salivary Repeated Wdgut Salivary Infestation Gland Infestation Gland ^ Pre-oviposition Period Oviposition Period 6.5 n b b 22
6 20 5.5 18 5 . 4.5 16 4 14 Repeated Midgut SaUvary Repeated Midgut Salivary Infestation Gland Infestation Gland
Non-Viable Eggs F Egg Incubation Period 14 22 21
« 20 . lis 18 17 m . Repeated wlSgui Salivary Repeated Hvadgm Salivary Gland Infestation Gland G Egg Mass Weight H Fecundity Efficiency Index 160 0.9 l É l b
iJLûJHRepeated widgut Salivary ; Repeated r midgut ii Salivary H Infestation Gland Infestation Gland
Figure 2.6: Fecundity parameters off?, sangu/neus females from individual infestations on dogs immunized with tick tissues. Dogs were immunized three times (intradermally) at 21 d intervals with tick salivary glands or midgut. The dogs were subjected to two challenge infestations, separated by a 21 d interval, with 80 female and 40 male ticks per infestation one week after the final immunization. Engorged, detached females were weighed and incubated separately to observe all fecundity performance parameters of each individual including the number of females that died after engorgement (A), the number of females that failed to oviposit (B), the pre- oviposition period between detachment of the females and the beginning of oviposition (C), the period required for females to oviposit (D), the number of females that oviposited non-viable eggs (E), the incubation period required for larvae to hatch from eggs from each female (F) and the weight of the egg mass from each female (G). The fecundity efficiency index (H) was calculated by dividing the engorged weight of each female by weight of the egg mass-produced by that female. Closed and open bars represent the first and second challenge infestations, respectively, for each panel displayed. Results are shown as Least Square (LS) Means ± Standard Error; different superscript letters in each panel indicate significantly different LS Means (p < 0.05). 71 CHAPTER 3
HUMORAL IMMUNE RESPONSE IN DOGS PREVIOUSLY IMMUNIZED
WITH SALIVARY GLAND, MIDGUT, OR REPEATED INFESTATIONS WITH
RHIPICEPHALUS SANGUINEUS
INTRODUCTION
It has been demonstrated that host immune responses to Ixodid ticks
acquired through either tick infestation or artificial immunization with tick
antigens have severe effects on tick feeding, reproduction and survival
(reviewed by Willadsen, 1980; Wikel, 1982, 1984; Wikel and Allen, 1982;
Brown, 1985; Barriga, 1994). The mechanisms implicated in the expression of
this immunity include antibody, cell mediated immunity and soluble mediators
including histamine and complement (Ribeiro et al. 1985). Humoral antibody
have been shown by many investigators important in the host defense
mechanism (Wikel and Allen, 1976; Robert and Kerr, 1976; Ackerman et al.
1981).
There is evidence that immunomodulation is a common phenomenon of tick infestation (Wikel et al. 1994; Wikel, 1996; Barriga et al. 1993). 72 The immunosuppressive effect of tick infestations may play an important role in evading host defense system. This effect might lead to the question as to why infestations of R. sanguineus do not induce acquired resistance in dogs.
The altered antibody level from immunization with salivary gland or midgut antigen compared to the antibody titer of infestation group should demonstrate this effect. The ELISA has been extensively used as an immunological tool of antibody investigations in either the diagnosis or analysis of various parasites
(De Lello and Boulard, 1990; Colwell and Baron, 1990; Hollanders et al. 1997;
Machado et al. 1997). Hernandez et al. (1995) reported that ELISA technique was a highly efficient detector of anti-R sanguineus antibodies and this technique is used frequently to investigate serological reactions against other tick antigens.
In this present study, antibody patterns of three groups of dogs were examined; the first and second group were immunized by tick salivary gland and midgut antigen respectively, and the last group was repeatedly infested by adult R sanguineus (control group). Our first hypothesis for this study was that
TSG- or TMG-lmmunization draws different antibody reactions from repeated infestations of Rhipicephalus sanguineus. To test this hypothesis, an ELISA was used to measure and compare the dynamics of those dog sera. The second hypothesis was immunomodulatory phenomena occurred during infestation and affected on the dynamics of antibody responses.
Immunosuppressive effects of challenged infestations in immunization groups were evaluated by a comparison of antibody profiles with infestations in the 73 control group. The last hypothesis was different stages or internal tissues of R. sanguineus contained immunogens that were related with salivary gland or midgut origin. The ELISA was also used to determine if an antibody response generated after immunization or infestations recognized either adult stages or internal tissues of this tick.
74 Materials and Methods
Ticks. Adult R. sanguineus were obtained from the Medical Entomology
Laboratory, the Oklahoma State University, and maintained at 30 °C with a 12-
h photoperiod and 90% relative humidity. Separately engorged females were
kept in glass tubes throughout oviposition, and eggs were stored separately
until hatching.
Antigen Preparation.Tick salivary gland antigen was prepared from male
and female R. sanguineus that had fed 3 to 5 day on a guinea pig. Ticks were
placed into 0.15 M phosphate buffered saline, pH 7.4 (PBS) and opened along
their dorsal surface. Salivary glands were removed, dissected free of other
tissues, and placed into PBS at 4 °C. Salivary glands, collected from 25 adult
ticks (15 females and 10 males), were suspended in 1% SDS and 5%
2-mercaptoethanol (2-ME), incubated in a water bath at 56 °C overnight and
boiled 5 minutes. The solution was cooled to room temperature, transferred
into a 12,000-14,000 molecular weight cut off dialysis tube (Spectra/Por7,
Denver, CO), immersed into 1 liter PBS, and left at 4° 0 on magnetic stir
overnight (PBS was changed every 4-6 h). The dialyzed mixture was filtered
and mixed with an equal volume of Freund's complete or incomplete adjuvant
H37Ra (Difco laboratories, Detroit, Ml) to a final volume of 1 ml.
Tick midgut samples were also removed at 4 °C in PBS. These organs were disrupted for 30 s in PBS at 4° 0 with a tissue homogenizer followed
sonication for 15 s (Model 300 Sonic Dismembrator, Fisher Scientific,
75 Pittsburgh, PA), set at 35% and 60% output power, a total often times. The homogenates were dialyzed in PBS at 4 °C ovemight and centrifuged at
16,000 G for 30 min at 4° C. Both TSG and TMG preparations were sterilized by filtration through a 0.45 pm filter (Millipore, Bedford, MA) and mixed thoroughly by sonication with the Freund's adjuvant to a final volume of 1ml prior to immunization.
Experimental Hosts. Nine Beagle dogs were purchased from HRP
(Kalamazoo, Ml) for this experiment. All dogs were 8-month-old females that weighed 6-8 kg. Dogs were randomly assigned to one of the three groups, immunization of Dogs. Experimental group 1 was inoculated intradermally with the TSG preparation, and group 2 was inoculated intradermally with the
TMG preparations three times at 21-d intervals. Each dog received 250-450 pg/ml of TSG or TMG mixed with Freund's complete adjuvant for the initial immunization or incomplete adjuvant for the second and third immunization.
The third experimental group (control group) was infested five times at 21-d intervals.
Infestation.All infestations consisted of 80 female and 40 male unfed adult ticks per dog. Dogs immunized with TSG or TMG were submitted to challenge infestations seven days after the last immunization, and a second time 21 day later.
Detection of antibodiesSera from dogs immunized with salivary gland or midgut or repeated infestation was tested by ELISA against antigen extracts of
76 eggmass, unfed larva, fed larva, nymph, muscle, nerve, reproductive, midgut,
and salivary glands. The ELISA used for the detection of anti-tick antibodies
was a modification of that of Barriga (1990). Briefly, the 96 well microtitre
plates were coated overnight at 4° C by the addition of 3pg of each antigen
diluted in a carbonate coating buffer to 0.1 M at pH 9.6. After washing five
times with 0.05 % PBS-Tween20, the postcoating was done by the addition of
100 |il per well of 0.1% BSA in PBS and incubated for 2 hours at room
temperature. Once the plates were washed, 100 jal per well of sample sera
from dogs (1: 100) were added to each well and incubated for 2 hours at room
temperature. After washing, 100 pi per well of peroxidase-conjugated goat
immunoglobulins to dog immunoglobulins (ICN, Aurora, OH) at 1: 2500 were
added to each well and incubated at room temperature for another 2 hours.
After a final wash, 100 pi per well of peroxidase substrate (2, 2'-azino-di- [3- ethylbenzthiazoline sulfonate]) (ICN, Aurora, OH) at 0.05 % concentration and
0.03% hydrogen peroxide were added. An ELISA reader was set at 405 nm to read the plates. When the color started to develop, we began reading the plates. We stopped when optical densities reached 2.5 or at 60 minutes of the reaction. Simultaneously, a positive and negative control without antigen, primary antibody, secondary antibody, and substrate were read to ensure that the calorimetric reaction was due to the formation of the antigen-antibody complex and not to non-specific reactions.
77 Results
Acquisition of resistance by immunization with tick salivary gland
or midgut. The feeding and fecundity performance of female R. sanguineus
ticks fed on dogs previously exposed to repeated infestations or immunized
with TMG or TSG were previously determined (Jittapalapong, 1999). Briefly,
ticks fed on TSG-immunized dogs showed an increased duration of feeding
and significant reduction in engorgement weights and engorgement numbers
as compared with ticks fed on control dogs (P<0.05). The egg mass weights
also dropped for ticks fed on TMG-immunized dogs (P<0.05). However, egg
mass weights did not drop significantly for ticks fed on dogs immunized with
TSG. The oviposition period increased for females fed on dogs immunized with either TMG or TSG. No significant differences were determined among the three groups for mortality, failure to oviposit, and egg viability. The tick performance values are summarized in Table 3.1.
Antibody response of dogs exposed to immunization with TMG,
TSG, or repeated infestations.Generally, anti-TMG antibody levels peaked after the second immunization then leveled off or gradually declined, while anti-TSG levels increased gradually and then declined after the first challenge infestation. The highest level of anti-TSG antibodies recognized egg mass antigen after the third immunization and gradually declined, there were moderate levels of anti-TMG antibodies binding to egg mass antigens, and no reactions were observed in repeated infestation dogs (Fig. 3.1a). Anti-TSG
78 antibodies recognized egg mass antigens at greater levels anti-TMG
antibodies.
A similar response was observed with unfed larval antigen (Fig. 3.1b).
The antibody response trends were similar for egg mass and unfed larvae
antigens, and sera from TSG-immunized dogs contained the highest level of
antibodies recognizing the unfed larvae antigen. Sera from dogs exposed only
to repeated infestations had the lowest levels of antibodies to all antigens
tested. Sera from TMG-immunized dogs contained the highest antibody levels
recognizing the fed larvae antigen (Fig. 3.1c). However, no recognition of fed
larvae antigen was detected in repeated infestation sera. Dogs immunized
with TSG or TMG displayed similar responses to nymph antigen, and lower
levels of antibodies recognizing nymph antigen were detected in the repeated
infestation group (Fig. 3.Id).
Sera from TSG-immunized dogs contained higher antibody levels to
TMG than that of TMG-immunized dogs (Fig.3.1e). Repeated infestation
resulted in a slight increase of levels of antibodies binding to midgut antigen,
but these levels did not significantly increase after the first infestation.
Sera from TSG and TMG immunized dogs responded differently to salivary
gland antigens. Anti-TSG sera recognition of salivary gland antigen gradually
increased from the first immunization to the first challenge infestation, and
dropped after the second challenge infestation (Fig. 3.If). Anti-TMG sera
displayed a similar pattern against salivary gland antigen, but at lower levels than those observed with TSG-sera. Antibodies to TMG were the highest after 79 the first challenge infestation, then declined after the second challenge
infestation. In the repeated infestation sera, antibody responses to salivary gland antigens continuously increased after the first infestation to their highest level after the fourth infestation and then slightly dropped.
DISCUSSION
Host immunoglobulins have been implicated as important effectors for acquired resistance to tick infestation (Brossard, 1979; Askenase, 1982).
Resistance to infestation with Boophilus microplus was passively transferred to naïve cattle by bovine immune serum (Roberts, 1976). Similar work demonstrated that the degree of resistance to Ixodes ricinus in rabbits,
(Brossard, 1979) and Ambiyomma americanum, R. sanguineus, and
R. appendiculatus in guinea pigs, (Askenase, 1982; Brown, 1985; Gill, 1987) was correlated to IgG titer.
The present study demonstrated that immunization of dogs with TMG or
TSG induced humoral immune responses to tick antigens found in both midgut and salivary gland in addition to immature tick stages, while repeated infestation induced little response. The higher level of anti-TSG IgG following successive immunizations corresponded with the previously reported higher level of resistance, (Jittapalapong, 1999) indicating that IgG titer is associated with canine resistance to infestation with R. sanguineus. Antibodies to TSG might contribute to resistance by neutralizing salivary components vital to tick
80 feeding. The level of anti-TSG IgG was positively associated with the level of
resistance (Jittapalapong, 1999).
These results indicated that R. sanguineus TMG and TSG were highly
immunogenic, and induced production of antibodies that recognized all four
tick development stages. These antibodies were also highly cross-reactive
between TMG and TSG antigen preparations. Bm86, which is the component
of a commercial vaccine against S. microplus, is of tick midgut origin
[Willadsen, 1989; Willadsen, 1989; Willadsen, 1992; Willadsen, 1995;
Willadsen, 1997). This vaccine was designed based on the concept that such
"novel" or "concealed" midgut antigens, which are not presented to the host immune system during natural tick feeding, can invoke an immune response that will damage ticks after uptake of host blood containing midgut-specific antibodies along with other humoral effectors such as complement. A similar mechanism may have been responsible for the affect of the TMG and the cross-stimulatory TSG extracts on tick feeding in this study. Interestingly, both antigen preparations reduced tick fecundity, but through different means
(Jittapalapong, 1999). Ticks fed on TMG-immunized dogs produced reduced egg mass weights while those fed on TSG-immunized dogs suffered lower engorgement weights. In addition, TMG and TSG induced the production of antibodies that recognized immature tick stages and adult tissues. Thus, it may be possible to stimulate an immune response capable of reducing both tick fecundity and feeding of all three parasitic stages.
81 Repeated infestation resulted in the lowest levels of resistance to R. sanguineus, (Jittapalapong, 1999) and the lowest levels of antibodies to all antigen sources tested. This confirmed other investigations where dogs failed to develop resistance against tick infestations (Chabaud, 1950; Ferreira et al.
1995; Theis, 1974; Bechara, 1994; Szabo, 1995). These results are not surprising since R. sanguineus is well adapted to its canine host in several ways. Osmoregulation is an important function of saliva in the tick, but components of this saliva also serve as immunomodulators (Ribeiro et al.
1985; Ribeiro and Spielman, 1986; Ribeiro 1987; Ribeiro, 1987). In addition, the locale of tick saliva at the host-parasite interface suggests that its components may be subject to selection for other immune evasion mechanisms such as molecular mimicry (Damian 1997). Finally, saliva released during natural feeding is likely to have a different composition, both quantitatively and qualitatively, than the TMG and TSG antigen extracts used in this work. Taken together, these considerations indicate salivary components released during tick feeding are not as likely to invoke protective immunity.
82 References
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86 Treat Number of Weight of Weight of Egg Feeding Oviposition
m ent Engorgement Engorged mass Period Period
(Number of Female (mg ± SE) (Days ± SE) (Days ± SE)
ticks ± SE) (mg ± SE)
TMG 33.67 ±3.39“ 214.36 ±4.62“ 86.46 ± 4.70“ 8.64 ±0.11“ 18.67 ±0.22^
TSG 32.83 ± 3.39“ 174.66 ±5.09^^ 113.31 ±4.37“ 9.55 ±0.08“ 18.20 ±0.21“
INF 51.20 ±2.14“ 204.06 ± 2.33“ 114.53 ±2.19“ 8.42 ± 0.07“ 16.61 ±0.11''
TABLE 3.1; Feeding and fecundity performance of female Rhipicephalus sanguineus ticks fed either on dogs that had been immunized with tick midgut (TMG) or salivary gland (TSG) and challenge infestations, or repeatedly infested (INF) with adult male and female of R. sanguineus. Results are shown as Least Square (LS) Means ± Standard Error (SE) superscript indicate a significant difference between values within each parameter as determined by Tukey-Kremer method Least Square Mean with statistical difference (p value < 0.05)
87 ■ TSG d □ IMG 2 - □ TIMG 0INF 1.5 H
■ TSG e □ TMG □ TMG
■ TSG f □ TMG □ TMG
Figure 3.1: Humoral responses of dogs previously immunized with tick salivary gland (TSG) or midgut (TMG) or repeatedly infested (INF) were tested by ELISA to egg mass (a), unfed larva (b). fed larva (c). nymph (d), midgut (e), and salivary gland (f) antigen. In TSG or TMG group, dogs were immunized three times and challenge twice infestation by 80 female and 40 male ticks per infestation at 21-day intervals. In the repeated infestation group, dogs were infested five times with the same number of ticks. The series of sera (x-axis) of dogs including pre-immune (1), after the first immunization or infestation (2), after the second immunization or infestation (3), after the third immunization or infestation (4), after the first challenge infestation or the fourth infestation (5), and after the second challenge infestation or the fifth infestation (6) were collected and performed ELISA and their results were measured at OD 405 (y- axis). 88 CHAPTER 4
TICK SALIVARY GLAND PROTEINS RECOGNIZED BY SERA OF
RESISTANT DOGS
INTRODUCTION
Acquired resistance was studied in Rhipicephalus sanguineus by Garin
and Grabarev (1972), who found that rabbits immunized with R. sanguineus salivary gland extract were resistant to infestation with the adult stage of the tick. However, this resistance did not appear to be greater than that induced by repeated infestation of rabbits (Schorderet & Brossard, 1993).
There is evidence that resistance to tick infestation is accompanied by the production of anti-tick antibodies that have been detected in the sera of repeatedly infested or immunized hosts. Njau & Nyindo (1987), using an
ELISA technique found a high titer of anti-R appendiculatus antibodies associated with resistance due to repeated tick infestation of rabbits.
Askenase et al (1982) conferred protection against infestation with R appendiculatus and Ixodes holocyclus in guinea pigs by transferring resistant sera to naïve hosts. 89 Tick salivary gland secretions are a complex array of proteins and
mucins that facilitate tick feeding, through anticoagulant, anesthetic and
immunomodulatory effects (Ribeiro et al. 1985; Ribeiro and Spielman, 1986;
Ribeiro 1987; Ribeiro, 1987). The components of tick saliva can change
according to the host on which the tick is feeding (Wang & Nuttall 1994).
In contrast to other tick-host models, several reports have indicated that dogs do not develop acquired resistance to R. sanguineus ticks after multiple infestations (Chabaud, 1950; Theis and Budweiser, 1974; Bechara et al. 1994;
Szabo et al. 1995). Conversely, other host species such as rabbits, guinea pigs, sheep, and cattle have been shown to develop acquired resistance to R. sanguineus (Trager, 1939; Garin and Grabarev, 1972; Boese, 1974; Brown,
1985; Wikel, 1981; Barriga et al. 1991, 1995; Sahibi et al. 1997; Hernandez et al. 1994). One reason for the discrepancy between these hosts abilities to develop resistance to R. sanguineus may be due to some aspect of the canine immune response to the tick (e.g., the type of immune effectors invoked or the immunogens recognized). Another possibility is that R. sanguineus is better able to evade the immune response of its natural, canine host through processes such as immunodulation and molecular mimicry (Wikel 1997;
Damian 1998).
Resistance to infestation by R. sanguineus has been elicited in dogs by repeated infestation or immunization with tick midgut or salivary gland tissue extracts (Jittapalapong et al. 1999a). The repeated infestation group of three naïve dogs was infested five times at 21-d intervals by the same numbers of 90 ticks. In each Immunized group, three tick-naive dogs were immunized three times with TSG or TMG extract, and twice challenged by allowing 80 female and 40 male adult ticks to feed on each host. Resistance was measured by the observation of tick feeding and fecundity performance parameters.
Humoral responses were detected by ELISA and tested with salivary gland or other tick tissues (Jittapalapong et al. 1999b).
Identification of defined immunogenic molecules is an essential part of dissecting the events involved in acquisition and expression of resistance to ticks. An understanding of the antigens that invoke resistance may lead to an effective vaccine for dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been used to study changes in individual proteins of the salivary glands of female Ixodid ticks during tick feeding (Wang
& Nuttall 1994). Female tick salivary glands undergo substantial growth, differentiation and development during feeding, and both qualitative and quantitative differences in the protein profiles of salivary glands have been observed soon after tick attachment to a host (McSwain et al 1982; Gill et al
1986; Shapiro et al. 1986; Gordon & Allen 1987; Wang & Nuttall 1994;). SDS-
PAGE followed by immunoblotting has been used to probe tick tissue extracts with sera from animals that developed resistance to tick infestation following tick attachment or immunization with complex tick extracts (Brown, 1988). The first hypothesis tested in this study was that salivary gland immunogens ofR. sanguineus recognized by resistant sera changed with the physiological state of the ticks during the feeding period. Western blot was used to characterize 91 antigens from R. sanguineus salivary glands collected during different days of
feeding that were recognized by antibodies from resistant dogs. The second
hypothesis tested was that R. sanguineus salivary gland protein expression
changes when the ticks are fed on hosts that were previously exposed by
infestation or immunization with tick tissue extract. This hypothesis was tested
by Western blot analysis of R sanguineus salivary glands collected during
repeated infestations of the control group, with sera collected from TSG-
immunized dogs after the final immunization but prior to the first challenge
infestation. The third hypothesis tested was that these resistant dog sera
would recognize salivary gland antigens from other ixodid tick species as well.
Thus, salivary glands from A. americanum, A. cajennense, D. variabilis, and R.
sanguineus ticks were also analyzed by Western blotting.
MATERIALS AND METHODS
Ticks. Adult R. sanguineus and A. americanum, A. cajennense, and D.
variabilis were obtained from Medical Entomology Laboratory at Oklahoma
State University, and maintained at 30 °C with a 12-h photoperiod and 90%
relative humidity.
Antigen Preparation. Tick salivary gland (TSG) antigen was prepared from male and female R. sanguineus that had fed 3 to 5 days on a guinea pig and unfed female of A. americanum, A. cajennense, and D. variabilis. Ticks were placed into 0.15 M phosphate buffered saline, pH 7.4 (PBS) and opened along
92 their dorsal surface. Salivary glands were removed, dissected free of other tissues, and placed into PBS at 4 °C. Salivary glands, collected from 25 adult ticks (15 females and 10 males), were suspended in 1% SDS and 5% 2- ME
(2-mercaptoethanol), incubated in a water bath at 56°C overnight and boiled 5 minutes. The solution was cooled to room temperature and dialyzed overnight
(12,000-14,000 molecular weight cut off; Spectra/Por7, Denver, CO) in PBS at
4 °C; the PBS was changed every 4-6 h. Tick midgut (TMG) samples were also removed at 4 °C in PBS. These organs were disrupted for 30 s in PBS at
4 °C with a tissue homogenizer followed sonication for 15 s (Model 300 Sonic
Dismembrator, Fisher Scientific, Pittsburgh, PA), set at 35% and 60% output power, a total often times. The homogenates were dialyzed in PBS at 4 °C overnight and centrifuged at 16,000 G for 30 min at 4 °C. Both TSG and TMG preparations were sterilized with a 0.45 |am filter (Millipore, Bedford, MA) and mixed thoroughly by sonication with an equal volume of the Freund's complete or incomplete adjuvant H37Ra (Difco laboratories, Detroit, Ml) at a final volume of 1 ml prior to immunization.
Experimental Hosts. Nine Beagle dogs were purchased from HRP
(Kalamazoo, Ml) for this experiment. All dogs were 8-month-old females that weighed 6-8 kg.
Immunization of Dogs. Experimental groups 1 and 2 were inoculated intradermally with the TSG or TMG preparations, respectively, three times at
21-d intervals. Each dog received 250-450 pg/ml of TSG or TMG mixed with
93 Freund's complete adjuvant for the initial immunization or incomplete adjuvant
for the second and third immunization. The third experimental group was
infested five times at 21-d intervals.
Infestation.All infestations consisted of 80 female and 40 male unfed adult
ticks per dog. Dogs immunized with TSG or TMG were submitted to challenge
infestations seven days after the last immunization, and a second time 21 d
later.
SDS-PAGE analysisSodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting were performed as described by Stich et al. (1999), with the following modifications on 70 x 80 x
1.0-mm continuous polyacrylamide slabs using MiniProtean II cell (BioRad).
The gels consisted of 4-20% linear gradient separating gel. Sample were dissolved in 50 mM Tris-HCI buffer (pH 6.8) containing 2% SDS, 5% 2-ME,
20%glyceral and 0.02% bromophenol blue, then heated for 5 minutes in a boiling water bath. The reduced, denatured samples were diluted 1:1 in Tris-
Glycine electrode buffer (25mM Tris, 190 mM glycine; pH 8.3) containing 1%
SDS prior to loading on the gel. Electrophoresis was carried out for 1 hour and
15 minutes at a constant 200 volts in Tris-glycine electrode buffer containing
1% SDS. immunoblot analysisFollowing electrophoresis, proteins were immunoblotted at a constant 100 volts for 2 hours onto a 0.22 ^m pore size nitrocellulose membrane in a transfer cell (BioRad) with a continuous buffer
94 system (39 mM glycine, 48 mM Tris, 0.0375 SDS and 20% methanol; pH 8.0-
8.9). The membranes were washed with TBS containing 0.05% Tween 20
(TBS-T) and blocked 3 hours at room temperature with 3% gelatin in TBS-T.
Once washed, the membrane were incubated overnight with dog sera diluted
1:100 in antibody buffer (1% gelatin in TBS) at room temperature with shaking.
Membranes were washed five times for 10 min TBS-T and incubated 2 h in
Cappel peroxidase-conjugated goat-anti-dog IgG (whole molecule) (ICN,
Aurora, OH) diluted 1: 2,500. The membranes were washed again and
incubated in 0.5% diaminobenzidine dihydrocholide (DAB) and 0.3% H 2O2
until the bands were developed completely or the substrate turned dark and the blots were then washed with ddH 2 0 to stop the reaction.
RESULTS
1. Analysis of female salivary gland antigens during feeding
Generally, an increase in the abundance and complexity of salivary gland proteins recognized by resistant sera was observed as tick feeding progressed. When examined by immunoblotting using a-TSG, a-TMG,and a- repeated infestation sera, a-TSG detected unique salivary proteins of female at 450, 250, 210, 130, 125, 105, and 38 kDa during the feeding period (Figure
4.1 and Table 4.1).
a-TMG recognized at least 11-protein bands during infestations (230,
205, 185, 170, 160, 150, 70, 45, 30, 15 and 9 kDa). Nine of female salivary
95 protein bands at 100, 90, 85, 65, 50, 28, 22, 18 and 10 kDa were commonly
found when tested with a-TSG and a-TMG.
2. Analysis of female salivary gland antigens during repeated
infestations
During the first infestation, proteins at 450, 250, 210, 130, 125, 105,
and 38 kDa in the female tick salivary gland were recognized by a-TSG
antibodies (Figure 4.1 and Table 4.1). a-TSG associated with tick resistance
recognized salivary gland protein bands at 330, 280, 195, 105, 85, 65, 32, and
18 kDa during the second infestation, but did not bind to 450, 250, 210, 130,
125, and 38 kDa (Table 4.3). Likewise, several salivary proteins were
exclusively demonstrated in the third infestation (250, 210, 170, 125, 120, 105,
95, 85, 75, 65, 38, 32, 25, 18, and 7 kDa) (Table 4.5). At least nine protein
bands ranging 18 to 250 kDa appeared twice during all three infestations. At
least of three proteins (105, 210, and 250 kDa) were commonly present in
each feeding day during every infestation. Preimmune dog sera showed no
reactivity to the salivary gland antigens (data not shown).
Antibodies from TMG-immunized dogs recognized most protein bands
in the first infestation while detected a fewer bands in the second infestation
(Table 4.1, 4.3). Salivary proteins of 230, 185, 170, 80, 70, 60, and 30 kDa were demonstrated in most infestations with TMG-antibodies.
The number of salivary proteins commonly recognized by a-TSG or a-
TMG were the highest in the first infestation (9 bands) and gradually dropped
96 to 7 bands and 1 band in the second and third infestations, respectively, a-
TSG serum apparently was more specific as the number of infestations
increased, while a-TMG or a-repeated infestation sera recognized fewer
bands.
3. Analysis of male salivary gland antigens during infestations
The protein profiles of male tick salivary glands were compared with
those of female ticks over three consecutive infestation periods. During
feeding, the profiles of male salivary glands change in both structure and
function to obtain blood from host (Sauer et al. 1986;Sonenshine 1991). For
unfed ticks, the salivary gland protein profile of male ticks was similar to that of
females. Thereafter, the profile of male salivary proteins changed, and the
changes differed from those observed in female salivary proteins.
When examined by immunoblotting using a-TSG, a-TMG, and a-
repeated infestation sera, a-TSG detected unique salivary proteins of male at
330, 305, 230, 210, 125, 115, 95, 85, 38, and 10 kDa during the first infestation (Figure 4.1 and Table 4.2). These sera also demonstrated more bands in the second and third infestations (Figure 4.2, 4.3 and Table 4.4, 4.6).
At least 10 protein bands frequently appeared during two infestations.
a-TMG recognized at least 4-protein bands during consecutive infestations. 70 kDa was uniquely found in every infestation. The number of male salivary protein bands that were commonly found between a-TSG and a-
TMG were less than those of female salivary glands.
97 4. Identification of salivary gland antigens ofsanguineus R compared to the other tick species
We determined the extent of cross-reactivity of antigens associated with induced immunity in several genera of ixodid ticks those infested dogs.
Results indicated that there is considerable cross-reactivity between salivary gland antigens and between the tick species tested, but that variation exists in the level of expression of these antigens.
a-TSG recognized bands in salivary glands from all the tick species examined. Salivary gland protein components from A. americanum,
A. cajennense, and D. variabilis were recognized by immunoglobulin from dogs that had been experimentally immunized three times with TSG of R. sanguineus. The reactivity profile of a-TSG antibodies with salivary glands from A. americanum and A. cajennense were not identical. a-TSG recognized salivary gland proteins of >A. americanum at 140, and 130 kDa. However, a-
TSG recognized more bands in A. cajennense than in A. americanum, specifically at 305, 230, 170, 150, 105, 85, and 65 kDa (Figure 4.4 and Table
4.7). The reactivity profile of a-TSG antibodies with salivary glands from D. variabilis was shown at 330, 285, 230, 210, 160, 105, 85, 70, and 60 kDa. a-
TSG identified salivary gland antigens at 205, 75, 55, 45, 40, 35, 30, 28, 25, and 22 kDa in all tick species examined (Table 4.7).
a-TMG antibodies demonstrated cross-antigenic proteins between
98 salivary gland and midgut tissues. a-TMG recognized salivary gland proteins
at 195, 55, 45, and 9 kDa found in the different tick species. In the repeatedly
infested group, these sera showed the lowest response to 55, 40, 30, 25, 22
and 9 kDa salivary gland in all tick species tested.
Discussion
One purpose of this study was to compare tick salivary gland antigens
expressed during the course adult tick feeding and recognized by sera from
resistant dogs. Tick salivary glands were characterized because they
appeared to be the source of many tick antigens recognized by immune
serum. The results from Western blots of salivary gland extracts taken from
ticks at different times throughout their feeding process revealed that not only
did a large number of salivary gland antigen exist, but also several of the
antigens varied markedly in concentration as the ticks fed.
Other investigators have tried to identify the specific tick antigen that
induces host resistance. Our Western blot analysis demonstrated nine TSG
bands, ranging from 10 kDa to 100 kDa, which were recognized by sera from
resistant dogs immunized with either TSG or TMG (Table 4.1 A). Seven protein bands at 450, 250, 210, 130, 125, 105, and 38 kDa, were uniquely recognized by TSG-immunized dog sera (Table 4.IB). The presence and concentration of some of the TSG antigens varied in relation to the tick-feeding phase.
Substantial changes in the profiles of protein antigens recognized in tick salivary glands during the course of feeding were observed. These changes
99 are probably associated with the alteration of structure and function of salivary
glands during feeding (Fawcett et al 1981). It was not surprising to find a
difference in the number of recognized polypeptides from female salivary
gland extract versus male salivary gland material after probing with anti-tick
sera. There is not only a basic difference in the feeding behavior between female and male ticks, but also in the morphology and physiology of the salivary glands as well. Female ixodid ticks feed for 3-4 days and then stop until mated. Once mated, females resume feeding and become replete with blood within 5-7 days, having increased in weight by nearly as much as 300 folds (Sauer 1977). While waiting to be mated, the female is in a state of apparent arrested feeding, but the salivary glands continue to be active and the tick probably feeds at a low level to maintain basic metabolic needs (Sauer
1977, 1979). Male ixodid ticks, however, generally take multiple, intermittent blood meals for 2-4 days and increase in weight by not more than two folds
(Sauer 1977). In addition, the salivary gland of the male is histologically distinct from the female (Sauer 1977). Therefore the acquisition of a large blood meal is crucial only to female ticks, and, they have developed the physiology for the longer feeding period required to obtain the required blood meal for efficient egg production (Diehl et al. 1982). The less pronounced changes observed in male tick salivary glands as compared to those of females were probably associated with the smaller bloodmeal requirements of male ixodid ticks to complete spermatogenesis (Oliver 1982). In addition, male ixodid salivary glands have a fourth acinus type that is not found in females 100 (Sauer et al 1986). It would be interesting to determine what salivary gland
acinus and cell types contain antigens that induce host resistance.
Salivary gland proteins appear to be conserved among the ixodid ticks
as evidenced by the ability of a-/\. americanum antibodies to recognize a great
number of proteins from D. variabilis and B. microplus tick (Brown 1988;
Brown and Askenase, 1981; Heller-Haupt et al. 1981; McTier et al. 1981).
Wheeler et al. (1991) found that hyperimmune serum to /. dammlni (I.
scapuiaris) from rats and rabbits contained antibodies to a large number of
antigens of a glycoprotein nature in salivary gland homogenates from adult ticks which cross-reacted with antigens from D. variabilis. Results of this experiment have shown that a-TSG or a-TMG of R. sanguineus recognized at
least 12 common protein bands from 9 to 205 kDa among A. americanum, A. cajennense, D. variabilis, and R. sanguineus (Table 4.7a). This indicated that there is a cross reactivity among ixodid tick species. There was also a unique group of bands that was found in each specie (Table 4.7b). This indicated that there is a cross reactivity among ixodid tick species. The observance of conserved protein bands among all four-tick species tested indicated that immunogens shared among ixodid ticks and may be of importance for the development of a cross-protective anti-tick vaccine.
A previous investigation indicated that immunization with TSG reduced female weight at engorgement, while immunization with TMG reduced the egg mass weights produced by challenge fed female ticks (Jittapalapong et al.
101 1999a). These results indicated that antibodies specific to TSG might be associated with reduced female body weight at engorgement, an important feeding performance parameter that also effects the tick fecundity index. The a-TSG sera used in this study was collected after the final immunization prior and to challenge infestation, when the highest degree of host resistance to infestation with R. sanguineus was found. This work represents the first essential step to characterize tick antigens that are associated with artificial induction of reduced tick performance. Immunization of dogs with purified antigens associated with a later reduced tick feeding and fecundity performances may result in a more pronounced resistance to R. sanguineus infestation.
References
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105 1 2 3 4 5 6 7 8 9
5 3
O4J0S '-’Î3S a æ m x is iS Bja sss 1 VR
njTT >u> SS
M.W. 1 2 3 4 5 6 7 8 9 (kOa) 5un """ 204 - 121 r 78
39.5
30.7
19.7 7.7
M.W. 1 6 7 (kOa) i M j n n x m
204 .
121 f
78
39.5 30.7 , ' t*a»i ta^n iw»» I7JM t7^ *'•»** 19.7 \ . 7.7 Ef
Figure 4.1: Western blot results of male and female salivary gland protein profiles during the first infestation of R. sanguineus recognized by a-TSG (A), a- TMG (B), and a-repeated infestation (0) dog sera (lane 1 = unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day female).
1 0 6 M.W. % ^ .1 (kDa)
204
121 78
39.5 30.7
19.7 7.7
M.W. (kDa)
204 121
78
39.5
30.7
19.7 7.7
M.W. (kDa) 204
121
78
39.5
30.7
19.7 7.7
Figure 4.2: Western blot results of male and female salivary gland protein profiles during the second infestation of R sanguineus recognized by a-TSG (A), a-TMG (B), and a-repeated infestation (C) dog sera (lane 1 = unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day female).
107 M.W.
wwsw#
M.W. _,A 2 3 456789 (kDa) "?r"' ■fj;r::- 204 121 78
39.5
, - . i ------
M.W. 8 (kDa)
204
121
78
39.5 30.7
19.7 7.7
Figure 4.3: Western blot results of male and female salivary gland protein profiles during the third infestation of R. sanguineus recognized by a-TSG (A), a- TMG (B), and a-repeated infestation (C) dog sera (lane 1 = unfed female; 2= fed 1 day male; 3= fed 3 day male; 4= fed 5 day male; 5= fed 7 day male; 6= fed 1 day female; 7= fed 3 day female; 8= fed 5 day female; 9= fed 7 day female).
1 0 8 M.W.
B
M.W. (kDa)
204 121 78 39.5
30.7 19.7
7.7
Figure 4.4: Western blot results of salivary gland protein profiles of different tick species recognized by a-TSG (A) and a-TMG (B) sera of dogs (lane 1= unfed male R. sanguineus] 2= fed male R. sanguineus] 3= unfed male A. americanum] 4= unfed female A. americanum] 5= unfed female A. cajennense] 6= unfed female D. variabilis] 7= unfed female R. sanguineus] 8= fed female R. sanguineus] 9= TSG vaccine antigen).
109 Tick Feeding Day 3 Antisera M.W. (KDa) IMG TSG INF IMG TSG INF IMG TSG INF IMG TSG INF IMG TSG INF Multiple Infestation Number 1 23 1 2 3 1 23 1 23 1 23 1 23 1 23 1 23 1 23 1 2 3 1 23 1 23 1 23 1 23 1 23 450 + --- — I— 330 + H— ■f “ + 305 + ---- + + " 280 —I— + 270 —(■ + 250 + 230 210 4-4- + ----- 205 4- 4— 4'4'4- — h — 4* — ' 195 185 170 _ — — .—- 160 150
130 4- - -
125 4------4----- 115 4----- 4* 4* “ “ 4- - - + ---- 4— 4* 4* — -I- — 4- ~ 4-
105 4. 4- 4. ^ ^ — 4- 44- 4 * ------4- — 4" — + ---- + 4- 4- 4-4-4- 4-44- 100 4 ------4- -
Table 4.1;Molecular weight ranged between 100-450 kDa of salivary gland proteins at different infestation immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R. sanguineus Tick Feeding Day
0 1 3 5 7 A n tis e ra M.W. (kD a) TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF Multiple Infestation Number 123 1 23 1 2 3 1 23 1 23 123 123 123 123 123 1 23 1 23 1 23 1 23 123 95 + + — - + + 4------(- + + 4 4— ----h — + + - + + — 4 4 4 4 4 — 90 ----1- + 4 - - 85 + ----f- H------H------4------4-4-4------4 - 4- ” + + ------4 4 4 4 4 4 ------+ + + 4 4 4 ------h 80 75 — ------4------h ------4------4 4 ------4 ------h —_ 70 - 4------h 65 + •" + + ----- + + + 4*----- 4- 4- — ----- 4- 4 4" — 4 - 4 4 ----- 4 ----- 4------+ + + 4 - 4 4 - 4 60 — 4— + - + 4— — ------h 55 + - + + ------f* — — — — — — — — 4- — — „ — “ — 4 ------4 — — — - 4 4 — k 4 50 ------+ - + 4------+ ----- 4------4 4 4* 4------4 4 - 4 ----- + + + 4 “ 4 4 4 4 45 4 ----- 4------38 — ------H------4------4 - 4 ------4------4------H— + 4 - 4 32 — 1— 4------+ — r — f* — 28 + - + H------f- 4-4-4- 4-4-4- — H 4- 4-4-4- 4 4 — -----4 4 4 4 4 4-4 ------H + + + 4 4 4 — 4 4 25 “ - 4 + + + ------22 ------— - -f + ------— ------4 4 4------4------h 4 + + + 4 ------4 - 4 18 + - + + ----- + — f- 4- “ - 4-4-4- 4 ----h - - 4- 4 4 4 4------h 4 - 4 4 4 4 4 — f- + + + 4 4 4 4 — 4 15 ------— — h ------4- 4------4 4 4 + + + —— -----— 10 -----+ + ------f- 4- " - 4- " ------4 4 — 4- — H ------H 4 4 - 4— 4 ------+ + + 4 4— ------f- 7 + ------■------4------— 4------4 —----- 4------4 —----- + H- + 4------h ------
Table 4.2; Molecular weight ranged between 7-95 kDa of salivary gland proteins at different infestations immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult males and females of R. sanguineus Different Infestations Antisera Infestation 1 Infestation 2 Infestation 3 Infestation 1,2, and 3 38; 105; 125; 130; 18; 38; 65; 85; 7; 18; 25; 32; 38; 38; 105; 210; 250 a-TS G 210; 250; 450 105; 195; 210; 65; 75; 85; 95; 250; 280; 330 105; 120; 125; 170; 210; 250
a-TM G 10;15; 32; 45; 60; 60; 70; 80; 170; 18; 22; 30; 60; 80; 60;170 150; 160; 170; 185; 230 170 185; 205; 230
a-IN F 22; 32; 305 28; 280; 305
a-TSG ; 10; 18; 22; 28; 50; 7; 10; 28; 90; 100 100 a-TM G 65; 90; 100; 190 100; 205
a-TSG ; 105; 305; 330 50; 330 18; 60; 85 a-IN F
a-TM G ; 45 60; 70 15; 22; 25; 65; a-lN F 115; 195
a-IN F; a - TMG; a- 18; 55; 65; 90; 28; 55; 90; 100; 7; 10; 50; 55; 55; 90; 115 115 115 90; 115; 330 TSG
Table 4.3: Summary of Western blot results of female R. sanguineus salivary gland proteins (kDa) at different Infestations recognized by sera of dogs that were either repeated Immunized by using salivary gland (TSG) or mIdgut (TMG) homogeneous antigen or multiple Infested (INF)
112 Different Tick Species A. americanum A. cajennense D. variabilisR. sanguineus Dog Antisera M.W. TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF (kDa) 330 305 285 270 250 230 210 205 195 185 170 150 130 125
110 105
Table 4.4: Molecular weight ranged between 105-450 kDa of unfed salivary gland proteins of different tick species Immunoblotted by sera of dogs that were either repeated Immunized by using salivary gland (TSG) or mIdgut (TMG) antigen or multiple Infested (INF) by adult females of R. sanguineus
113 Different Tick Species A. americanum A. cajennense D. variabilisR. sanguineus Dog Antisera M.W. TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF
100 95 90
75 70 65 60 55 50 45
32 28 25 22
Table 4.5; Molecular weight ranged between 10-100 kDa of unfed salivary gland proteins of different tick species immunoblotted by sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females of R. sanguineus
114 Different Tick Species Antisera A. americanum A. cajennense D. variabilis R. sanguineus
22; 32; 150; 205 10; 15; 32; 38; 75; 85; 18; 32; 45; 50; 60; 75; 32; 45; 55; 60; 70; 75; a-TSG 100; 205; 230 90; 125; 205; 210; 230; 110; 115; 125; 130; 185, 305;330 205; 210; 305; 330
a-TMG 115; 170; 195; 330 18; 195; 285 110 ; 195 85; 195; 285
a-INF 65; 125; 185; 270 95; 110 10; 15; 95; 185; 270 10; 50; 250; 270
LA a-TSG; 80; 130 25; 28; 45; 65; 80; 125; 25; 55; 70; 85; 170; 285 90; 105 a-TMG 150; 170; 185; 305
a-TSG; 25; 28; 38; 50; 100 90 22; 38; 105; 250 18; 25; 38; 95; 100 a-INF a-TMG; a-INF 10; 15; 250 70; 270
a-INF;a- TMG; a- 45; 55 50; 55;105 28 22; 28 TSG
Table 4.6: Western blot results of unfed salivary gland proteins (kDa) of different tick species recognized by sera of dog that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus CHAPTER 5
PROTEIN PROFILES OF DIFFERENT STAGES AND INTERNAL TISSUES
OF RHIPICEPHALUS SANGUINEUS RECOGNIZED BY SERA OF
RESISTANT DOGS
INTRODUCTION
Ectoparasites are a major worldwide problem for humans and animals
by debilitating agents themselves and as vectors of disease. It is well established that various species of ixodid ticks induce a degree of resistance
in the host and that resistance is immunologically mediated (Wikel and Allen,
1982). The nature of the immunological response following tick infestation varies with the tick-host model. Wikel (1984), Brown (1985), and Barriga
(1994) have comprehensively reviewed this aspect.
Dogs repeatedly infested with their natural tick Rhipicephalus sanguineus do not develop resistance even after years of exposure (Chabaud
1950; Theis and Budwiser 1974), whereas rabbits and guinea pigs develop strong resistance to this species of ectoparasites (Garin and Grabarev 1972).
The acquired resistance developed by these laboratory animals is reflected by
116 a decline in tick engorgement weight, eggs mass production, and reduced egg viability (Garin and Grabarev 1972; Szabo et al 1994).
The nature of the immune response of the resistant host, which is critical to impair tick progeny survival, has not been fully elucidated. Wikel and
Allen (1976) demonstrated that antibodies have a role in the resistance of guinea pigs by using cyclophosphamide dose that greatly depleted B cells and only slightly affected T cells. Fujisaki (1978) and Brown (1982) showed that antibodies implicated in the immune response of guinea pigs or rabbits resistance to ticks belong to the IgG class, specifically the IgGI subclass, and that they work through cells with Fc receptors (Brown and Askenase 1985).
Experiments using transfer of immune serum from tick-resistant animals to susceptible animals reinforced the importance of antibodies and serum factors in improving resistance (Trager 1939; Brown 1982). The information about specific antigens involved in immune responses associated with acquired host resistance would be greatly necessary. Brown (1988), using the
Western blot technique in a guinea pig showed that sera from tick-immunized animals identified some proteins implicated in resistance.
Very little is known about the effects of antibodies on ticks that engorge on resistant hosts, or which tissues of the tick body are possibly immunogenic.
The objectives of this work were first to investigate whether immunized dogs with different tick tissues or repeatedly infested dogs recognized the same antigen. The other objective was to determine which antigens in different tick tissues or stages were recognized by resistant sera. 117 In the present study, we compared the ability of sera originating from resistant dogs collected before and after immunization with tick salivary gland
(TSG) or midgut (TMG) extracts and after repeated infestations. To identify tick antigens in SDS-PAGE, fractionated R. sanguineus tissues including midgut, muscle, nerve, reproductive, and salivary gland extract, and tick stages including eggmass, unfed larva, fed larva, and nymph extracts were immunoblotted by TMG or TSG-immunized sera. We have analyzed stage- specific and shared antigens derived from R. sanguineus using sera from dogs immunized with salivary gland or midgut and challenge infestation with adults of this tick.
118 MATERIALS AND METHODS
Ticks. Adult R. sanguineus were obtained from the Medical Entomology
Laboratory, Oklahoma State University, and maintained at 30 °C with a 12-h photoperiod and 90% relative humidity. Engorged females were kept individually in glass tubes throughout oviposition, and eggs were stored separately until hatching.
Antigen Preparation.Tick salivary gland and other tissues were prepared as described in chapter 4.
Experimental Hosts. Nine Beagle dogs were purchased from HRP
(Kalamazoo, Ml) for this experiment. All dogs were 8-month-old females that weighed 6-8 kg.
Immunization of Dogs. Experimental groups 1 and 2 were inoculated intradermally with the TSG or TMG preparations, respectively, three times at
21-d intervals. Each dog received 250-450 pg/ml of TSG or TMG mixed with
Freund's complete adjuvant for the initial immunization or incomplete adjuvant for the second and third immunization. The third experimental group was infested five times at 21-d intervals.
Infestation.All infestations consisted of 80 female and 40 male unfed adult ticks per dog. Dogs immunized with TSG or TMG were submitted to challenge infestations seven days after the last immunization, and a second time 21 d later.
119 SDS-PAGE Analysis. Sodium dodecylsuiphate polyacrylamide gel
electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970)
and revised by Barriga et al (1991) on 70 x 80 x 1.0-mm continuous
polyacrylamide slabs using MiniProtean II cell (BioRad). The gels consisted of
4-20% linear gradient separating gel. Sample was dissolved in 50 mM Tris-
HCI buffer (pH 6.8) containing 2% SDS, 20%glyceral, 0.02% bromophenol blue diluted 1:1 in Tris-Glycine electrode buffer (25mM Tris, 190 mM glycine; pH 8.3) containing 1% SDS. When reducing conditions were required, 5% 2-
Mercaptoethanol was added and the sample solution was then heated for 5 minutes in a boiling water bath. After loading each extract into their correspondent gel lane, electrophoresis was carried out for 1 hour and 15 minutes at a constant 200 volts in Tris-glycine electrode buffer containing 1%
SDS.
After staining gels in staining solution (40% methanol, 10% acetic acid,
0.25% Coomassie blue R-250) overnight, gels were immersed in destaining solution (40% methanol, 10% acetic acid) and placed on a shaking platform until the background was clear. Molecular weights were estimated using commercial MW markers (Kaleidoscope Prestained Standard Molecular
Weight, BioRad).
Immunoblotting analysis.Following electrophoresis, proteins were immunoblotted at a constant 100 volts for 2 hours onto a 0.22 pm pore size nitrocellulose membrane in a transfer cell (BioRad) with a continuous buffer
120 system (39 mM glycine, 48 mM Tris, 0.0375 SDS and 20% methanol; pH 8.0-
8.9). The membranes were washed with TBS-Tween 20 and were blocked 3
hours at room temperature on shaking by addition of 3% gelatin in TBS-Tween
20. Once the membrane were washed, the membrane was incubated in sample sera from dogs in antibody buffer (1% gelatin in TBS) in the proportion
1:100 at room temperature on shaking overnight. The next morning, this membrane was washed and incubated in peroxidase conjugated goat immunoglobulins to dog immunoglobulins at the 1: 2,500 dilution around 2 hours on a shaking platform. This gel was washed again and incubated in the substrate DAB at the 0.5% concentration and 3% H 2O2 until the bands were developed completely or the substrate turn dark. When bands developed sufficient color the blots were removed and washed with distilled water to stop the reaction.
121 RESULTS
ANALYSIS OF ANTIGENS USING ANTI-SALIVARY GLAND SERUM (TSG)
1. Analysis of antigens present in different tick development stages
Immunoblot analysis employing resistant dog serum was used to
ascertain the presence of tick antigens in other development stages of R.
sanguineus. Most of the major antigens in the adult salivary glands were
detectable in egg mass, unfed larvae, fed larvae, and nymphae. Protein bands
ranged from 10 to 190 kDa and are shown in figure 5.1 and summarized in
Table 5.1. Five shared proteins at molecular weights of 40, 45, 85, 95 and 105
kDa were present in all tick development stages. Five proteins at 10, 25, 35,
65, and 190 kDa were found only in unfed larva, fed larva, and nymph stages.
Nymph and unfed larva shared 15 proteins at the same molecular weight while
unfed and fed larva shared 11 proteins.
2. Analysis of antigens present in different tick tissues
Immunoblot analysis employing sera from the resistant dogs was used to characterize tick antigens from midgut, muscle, nerve, reproductive, and salivary gland antigens. Antibodies from TSG-immunized dogs recognized common protein bands at 65, 75, 95, and 105 kDa in all tick tissues. Most antigens found in salivary glands were not detectable in tick midgut. Resistant antibodies from the a-TSG-sera recognized two unique proteins at 80 and 125 kDa. Most of the antigens present in salivary gland also likely appeared in tick muscle (14 proteins) and reproductive antigens (12 proteins). Excluding
122 midgut, three proteins at 45, 85, and 130 kDa were present in muscle, nerve,
reproductive, and salivary gland. a-TSG sera also recognized unique muscle
and reproductive organ proteins at 270, 150 and 70 kDa (Table 5.4).
ANALYSIS OF ANTIGENS USING ANTI-MIDGUT SERUM (TMG)
1. Analysis of antigens present in different tick instars
The profiles of antigens recognized by a-TMG sera is illustrated in Fig
5.2 and Table 5.5. These sera recognized a group of unique proteins from egg mass, unfed-, and fed larvae, and nymphs at molecular weights of approximately 15, 95, and 190 kDa. Seven unique proteins (35, 55, 60, 70, 75,
115, and 140 kDa) were identified only in egg mass extracts, whereas 65 kDa were identified only from unfed-, fed larva and nymph extracts. a-TMG sera recognized the highest number of bands in egg mass extract (19 bands) compared to 10 bands in unfed larvae, 9 bands in fed larvae, and 8 bands in nymphs.
2. Analysis of antigens present in different tick tissues
The profile of antigens recognized by dogs’ sera (naïve, post immunization, and post-challenge infestation) is shown in Figure 5.2 and Table
5.5. Serum from artificially immunized dogs expressing tick resistance recognized approximately 8, 13, 5, 9, and 9 proteins from the midgut, muscle, nerve, reproductive, and salivary gland respectively. a-TMG antibodies recognized a 15-kDa protein in all tick tissues. This antibody also detected protein bands at 60, 50, 45, 32, 28, 22, and 10 kDa in midgut extract; the 22,
123 28, and 60 kDa were unique to the midgut antigen. a-TMG antibodies uniquely
recognized proteins at 35, 100, 110, 115, and 140 kDa in muscle, and at
proteins 40 and 105 kDa in the synganglion. Reproductive and salivary gland
tissues shared protein bands at 230 and 190 kDa. Proteins of 75 and 95 kDa
were common to muscle, nerve, reproductive, and salivary gland extract.
ANALYSIS OF ANTIGENS USING SERA FROM REPEATEDLY INFESTED
DOGS
1. Analysis of antigens present in different tick instars
Repeatedly infested dogs also expressed some resistance to infestation
with R. sanguineus. Sera from these dogs recognized ten conspicuous protein
bands in the egg mass (10, 15, 35. 45, 55, 75, 95, 105, 140, and 170 kDa),
seven in unfed larvae (10, 15, 70, 85, 95, 105, and 170 kDa), three in fed
larvae (10, 15, and 65 kDa), and none in nymph extract (Figure 5.3 and Table
5.3). Only two protein bands at 10 and 15 kDa were shared by egg mass,
unfed larvae, and fed larva extracts. The naïve dogs sera recognized no
bands.
2. Analysis of antigens present in different tick tissues
Sera from repeatedly infested dogs recognized five proteins in midgut
(80, 70, 60, 15, and 10 kDa), six in muscle (45, 80, 100, 290, 345, and 420
kDa), three in nerve (80, 100, and 105 kDa), ten in reproductive (10, 55, 80,
95, 100, 275, 345, 400, and 435 kDa) and salivary gland (345, 100, 95, 80, 70,
60, 55, 45, 15, and 10 kDa) extract (Table 5.6). This antibody demonstrated
124 proteins at molecular weight of in midgut whereas it shown at of proteins in
salivary gland. A protein at 80 kDa was commonly found among those tick tissues. Excluding midgut, a protein of 100 kDa was shown in the other tick tissue. Repeatedly infested sera uniquely recognized two proteins (290 and
420 kDa) of muscle, one protein (105 kDa) of nerve, three proteins (275, 400, and 435 kDa) of reproductive tissue, and one protein (55 kDa) of salivary gland antigen.
DISCUSSION
Host resistance to ticks is expressed by reduced engorgement weight, longer feeding periods, decreased ova production, inhibited molting, and increased egg and tick mortality. Immunization with salivary gland affected both feeding and fecundity performance parameters. These results indicated that immunization with either TMG or TSG influenced tick fecundity, but through different mechanisms. Immunization with TSG apparently reduced fecundity by interference with bloodmeal uptake, while immunization with TMG reduced bloodmeal conversion into egg mass. Therefore, these dogs have demonstrated the development of immune resistance to R. sanguineus.
Although the resistance observed did not result in complete mortality or reduction in fecundity, it was indicative of the potential to establish protective resistance in dogs.
In general, the antibodies resulting from immunization had a greater affinity for tick tissues (Almeida et al. 1994). Although the serum from animals 125 immunized with TSG recognized salivary gland components, it did not present stronger reactivity to these structures than other sera raised against their structures. In fact, the salivary glands were not the most reactive sites for any of these sera. In this experiment, muscle was the most recognizable tissue by anti-TSG sera and had been showed the high relation to the salivary gland
(Table 5.4).
Antigenic differences were detected by TSG- or TMG-immunized dog sera between different tick tissues. While tick salivary glands were the most source of the antigen detected, tick midgut contained some antigens in less quantity than was found in salivary glands. Fujisaki et al (1981) also demonstrated a high titer of antibodies against midgut antigens in rabbits after tick infestations. Potential midgut antigens may be introduced into the animal host by regurgitation during feeding (Kemp et al 1982; Brown et al 1986). It has been proved that some part of resistance to tick feeding could fraction by host antibody attack internally on midgut antigens (Trager 1939; Allen and
Humphreys 1979; Ackerman et al 1980). In this experiment, anti-TSG recognized midgut antigens at 60, 65, 75, 95, and 180 kDa while anti-TMG detected salivary gland antigens at 15, 45, 55, 65, 70, 75, 95, 180, and 190 kDa (Table 5.4 and 5.5). Sera from TSG and TMG immunizations responded differently to salivary gland antigens. Interestingly, the midgut apparently contains immunogens related to salivary glands. Likewise, salivary glands consisted of immunogens that v/ere recognized by anti-TMG antibody. This result indicated that related antigens between salivary gland and midgut might 126 be associated with some resistant performances including reduced the
number of engorgement, engorged and egg mass weight of dog against R. sanguineus that v/as described by Jittapaiapong et al (1999). Thus, it is possible that the multivalent vaccine consisted of high different immunogens might increase the extent of host resistance to this tick burden.
Our results also indicated that many of the antigens recognized in adult tick salivary gland are absent from egg mass (70, 115, and 160 kDa), unfed larval (35 and 160 kDa), fed larval (35, 115 and 160 kDa), and nymphal (35,
70 and115 kDa) ticks (Table 5.1 & 5.4). Our results were supported by the work of Hernandez in 1995. His results have shown 32 and 49.8 kDa protein bands in larva and nymph extract when performed Western blot using anti larva sera of rabbits previously infested with R. sanguineus. Brown (1988) observed by Western blots a protein at 38 kDa which was recognized in egg, larval and nymphal extracts of A. americanum using anti-larval serum. Fujisaki
(1981) also reported some adult tick antigen missing from extracts of larvae and nymphs. In addition, an antigen studied by Willadsen and Riding (1979) shown an absent of a protease inhibitor from nymphs and adults in Boophilus microplus (Willadsen and Riding 1979). Moreover, the work of Whelan
(Whelan et al 1984) revealed that the larval and ova antigen detected by antibodies from resistant guinea pigs differ considerably. It has long been observed that resistance to infestation induced by feeding ticks of one stage in
127 tick development does not always confer resistance to other tick instars
(Trager 1939).
Our studies indicated that many different immunogenic tick molecules are inoculated into the infested host animal. It is impossible that all of these antigens function in eliciting host resistance to infestation. The differences in specific antigens found in different tick tissues and instars suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens. It is not possible to conclude which of the many antigens recognized are functionally involved in tick resistance. Some of the numerous antigens described may function together as subunits of larger and complex molecules.
The purification and test of every prospective protein bands were necessary to elucidate the antigens inducing resistance.
Development of resistance due to successive infestations appeared to be less pronounced than direct immunization, but this is not surprising since the dog is the natural host to which R. sanguineus is highly adapted. The resistance manifested by repeated infestation affected both feeding and fecundity performances in a manner similar to that of the salivary gland immunization group since the majority of antigens released into the host during feeding are probably secretory products of the tick salivary glands.
The sera of tick-resistant hosts have been shown to contain antibodies that bind specifically to tick salivary gland and midgut tissues since there were evidences that passive transfer of immune sera and peritoneal exudate cells from tick-resistant hosts conferred resistance to tick feeding. In the resistant 128 host a prominent increasing of antibodies were detected and demonstrated their association with tick tissue immunogens. Specific antigens from tick salivary glands and midgut have been associated with protective immunity against different tick species, and these tissues have been proved to induce resistance to ticks in dogs.
In addition to identification of protective immunogens, mechanisms of manipulating the host immune system by this tick must also be considered.
These immunomodulation mechanisms seem to be the most efficient under natural conditions, when hosts seemingly possess little measurable resistance against tick infestation. This phenomenon may explain why reduced tick performance parameters in this investigation were often recovered in subsequent infestations.
It is well known that ticks are distributed throughout the world in all countries and different species of ticks are parasitic on many species of animal, either wild or domesticated. Ticks and the diseases they transmit to livestock are a major animal health problem in almost all developing countries.
Either through their direct or indirect effects on animals, ticks causes major economic losses in all pet animals and livestock industries particularly in cattle industries.
The traditional method of control of ticks is application of acaricide that was first introduced in the 1950s and continues today to be almost the only method of tick control throughout the world. The emergence of tick resistance
129 to acarlcides, the accumulation of these chemicals in the environment and their rising costs have resulted in searching into alternative methods of control based on the use of host defense mechanisms. The effect of immunization can be long lasting and without the complication of residues. Vaccines are environmentally safe and should be very target-species specific. In addition, arthropod resistance to vaccines through selective adaptation is less likely to occur. Vaccines can be used to reduce the amount of pesticide necessary for control of ectoparasite. A commercial vaccine against B. microplus, a highly adapted one-host tick of cattle, has been developed with the midgut associated immunogen Bm 86, and has been shown to reduce the engorgement weight and egg laying capacity of engorged female ticks. The success of the B. microplus vaccine indicated that it may be possible to induce immune protection against R. sanguineus in dogs as has been done with guinea pigs. Immune resistance to R. sanguineus may reduce the fecundity and subsequent burdens of the ectoparasite, resulting in less direct damage due to heavy infestation of the host and perhaps curtailing the transmission of tick-borne pathogens. The results of this research indicated that dogs are good candidate animals for the study development and practical application of an ixodid tick vaccine. These animals gradually develop a strong immunologically mediated tick rejection response. These findings suggested that immunological vaccination of dogs against R. sanguineus would pose a new alternative for long term control of tick populations and the spread of tick- borne diseases. 130 There are a few important considerations before making an anti-tick
vaccine. Vaccination with midgut tissue induced an obvious resistance mainly
as a great reduction of tick fecundity that had the most advantage on tick
population control. However, this vaccine is therefore unlikely to prevent
infection by tick-borne pathogen. The major role of a putative tick vaccine
would be to control tick infestation per se and thus reduce or abolish the need
to apply acaricides. The salivary gland vaccine is more effective in preventing
feeding and partially effective in depresses fecundity, as well as interference transmitting diseases. Excluding midgut and salivary gland protein, other tick tissues including muscle, synganglion, and reproductive might be considered
as candidate antigen if they are capable of inducing resistance against this tick. Apart from prevailing tissues, there were evidences of ectoparasite tissues used as prospective antigen such as thoracic muscles of stable flies,
Stomoxys calcitrans, were used to vaccinate rabbits. Both stable flies and tsetse flies {Glossina morsitans) fed on the immunized rabbits were killed and some survivors showed other effects of vaccination including paralysis of legs, interference in deposition of endocuticle, and reduced post-emergence growth.
This evidence has brought the concept of multivalent vaccine, which consists of more than one-tick tissue ingredients. This candidate vaccine will enable in preventing ticks from feeding, inhibiting tick fecundity, increasing tick mortality, and stopping transmitting diseases. An effective immune response directed preferentially against different stages of ticks would be of a supplementary benefit to the host. 131 References
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133 Ribeiro, J. M. C., Makoul, G.T., Levine, J., Robinson, D. R., and Spielman, A. 1985. Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammlnl. J. Exp. Med. 161: 332-344.
Ribeiro, J. M. C., and Spielman, A. 1986. Ixodes dammini: Salivary anaphylactoxin inactivating activity. Experimental Parasitology. 62: 292- 297.
Ribeiro, J. M. C. 1987. Ixodes dammini: Salivary anti-complement activity. Experimental Parasitology. 64: 347-353.
Ribeiro, J. M. C., 1987. Roles of saliva in blood feeding by arthropods. Ann. Rev. Entomol., 32; 463-478.
Schorderet, S., and Brossard, M. 1993. Changes in immunity to Ixodes ricinus by rabbits infested at different levels. Medical and Veterinary Entomology. 7: 186-192.
Szabo, M. P. J., Arantes, G. J., and Bechara, G. H. 1995. Immunological characterization of adult tick Rhipicephalus sanguineus (Latreille, 1806) antigens by western blot analysis using sera from infested or vaccinated dogs and guinea pigs. Rev. Bras. Parasitol. Vet. 4: 79-83.
Szabo, M. P. J., and Bechara, G. H. 1997. Immunization of dogs and guinea pigs against Rhipicephalus sanguineus ticks using gut extract. Veterinary Parasitology. 68: 283-294.
Theis, J. H., and Franti, 0. E. 1971. Changing infestation rates of Rhipicephalus sanguineus (Latreille) (Ixodidae) ticks on dogs on Singapore island, 1965-1966. J. Med. Entomol. 8: 23-28.
Theis, J. H., and Budwiser, P. D. 1974. Rhipicephalus sanguineus: Sequential histopathology at the host-arthropod interface. Experimental Parasitology. 36: 77-105.
Trager, W. 1939. Acquired immunity to ticks. The Journal of Parasitology. 25: 57-81.
Trager, W. 1939. Further observations on acquired immunity to the tick Dermacentor vanabilis say. The Journal of Parasitology. 25: 137-139.
Wikel, S. K. 1981. The induction of host resistance to tick infestation with a salivary gland antigen. American Journal of Tropical Medicine and Hygiene. 30: 284-288.
134 204.00
1Z1.00
78.000
39.500
30.700
19.700
7.700
204.000
121.000
78.000
30.700
19.700
7,700
STD.MW.
2O4,000J5wmammm
3 9 , 5 0 0 : Y % : : : " '; '.
30.700"
19.700 7.700
Figure 5.1: Western blot analysis of different stages and tissues of R. sanguineus immunoblotted with different anti-TSG-sera; after the last immunization (A), after the first challenge infestation (B), and after the second challenge infestation (C). (Lane 1=egg mass; 2=unfed larvae; 3=fed larvae; 4=nymphs; 5 = midgut; 6 = reproductive; 7= muscle; 8 = nerve, and 9 = salivary gland extract)
135 Figure 5.2: Western blot analysis of different stages and tissues of R. sanguineus immunoblotted with different anti-TMG-sera; after the last immunization (A), after the first challenge infestation (B), and after the second challenge infestation (C). (Lane 1 = nerve; 2 = muscle; 3 = reproductive; 4 = midgut; 5 = salivary gland; 6 = nymphs; 7 = fed larvae; 8=unfed larvae, and 9 = egg mass).
136 ##
4
m
,ifL|y?f^-"'!M !% !‘
Figure 5,3: Western blot analysis of different stages and tissues of R. sanguineus immunoblotted with different a-repeated infestation sera; after the third infestation (A); after the fourth infestation (B); and after the fifth infestation (C). (Lane 1 = nerve; 2 = muscle; 3 = reproductive; 4 = midgut; 5 = salivary gland; 6 = nymphs; 7 = fed larvae; 8 = unfed larvae, and 9 = egg mass).
137 Tick Development Stages Egg Mass Unfed Larvae Fed Larvae Nymphs M.W. (kDa) Dog Antisera ‘TMGTSGINFTMGTSG INF TMG TSG INFTMG TSG INF 185 + 4 - 4 - 4 - 4 - 4 - 4 -
160 + + + 4 - 4 - 4 - 4 - 4 - 150 + + +
125 + + 4 - 4 - 4 - 115 + + 4 - 4 - 105 + + 4 - 4 - 4 - 4 - 4 - 95 + -f- + 4 - + 4 - 4 - 4 - 4 - 4 -
85 + + 4 - 4 - 4 - 4 - 4 - 4 -
75 4 - 4 - 4 - 4 -
70 + + 4 - 4 -
65 4 - 4 - 4 - 4 - 4 - 4 - 4 - 60 4 -
55 + 4 - 4 - 4 -
45 + -t- 4- 4- 4- 4- 4 -
38 +- + 4 - + 4 - 4 - 4 -
28 + 4 - 4 - 4 - 4 - 4 - 4 - 22 + + 4 - 4 - 4 - 4 -
18 4 - 4 -
15 + 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 -
10 + 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 -
7 4 - 4 -
Table 5.1: Western blot results of tick developmental stage proteins including egg mass, unfed larvae, fed larvae, and nymphs recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus
138 Tick Development Stage Proteins Recognized Antisera Egg Mass Unfed Larvae Fed Larvae Nymphs
7; 18; 28; 55; 75 22; 45; 85; 105; 7; 10; 18; 38; 45; a-TSG 115 55; 60; 70; 75; 115; 125
a-TMG 22; 60; 185
a-INF 70
a-TSG & 38; 70; 85; 115; 22; 38; 45; 65; 28; 38; 95; 125; 15; 22; 28; 65; a-TMG 125 185 160; 185 85; 95; 105; 185
a-TSG & 105 a-INF
a-TMG & 10; 15; 28; 55; 15 a-INF 75 a-INF & a-TMG & 45; 95; 105; 10; 15; 85; 95; 10; 65 a-TSG 150 160 160
Table 5.2; Western blot results of developmental stages of R. sanguineus recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen, or multiple infested (INF) by adult females and males of R. sanguineus
139 Tick Tissues Midgut Muscle Nerve Reproductive Salivary Gland M.W. Dog Antisera (kDa) TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF 420 400 345 325 290 270 230 205 O 185 170 150 130 115 105 100
Table 5.3: Molecular weight of tick tissue proteins ranged between 95-420 kDa recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus Tick Tissues
Midgut Muscle Nerve Reproductive Salivary Gland M.W. Dog Antisera
TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF TMG TSG INF 85 + + + + 80 + + + + + + 75 + + + + + + + + + 70 + + + + + 65 + + + + + + + 60 + + + + + + + 50 + + + + + + 45 + + + + + + + + + 38 + + + 32 + + + + 28 + + + + 22 + + + + 18 + + + + + 15 + + + + + + H- + + + 10 + + + + + + + + + 7 + + -i-
Table 5.4: Molecular weight of tick tissue proteins ranged between 7-85 kDa recognized by different sera of dogs that were either repeated immunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus Tick Tissue Proteins Antisera MIdgut Muscle Nerve Reproductive Salivary Gland
65; 75; 95; 105; 185 7; 22; 28; 38; 50; 45; 65; 85; 115;130 7; 22; 28; 38; 50; 7; 18; 22; 28; 32; a-TSG 65; 70; 85; 130; 65; 85; 105; 115; 38; 85; 105; 115; 205; 270; 325 130; 150; 185; 205 130; 325
a-TMG 22; 28; 32; 45; 50 150;170 15; 38 70; 170; 230
a-INF 70; 80 80; 290; 345; 420 80; 100 80; 100; 270; 345; 100; 345 400; 420 a-TSG; a-TMG 10; 15; 18; 32; 60; 75; 95 15; 18; 45; 75 65; 75; 185; 205 75; 95; 105; 115 a-TSG; a-INF 45 60 10; 60; 80
a-TMG; a-INF 10; 15 100 70
a-INF; a-TMG; 60 105 10; 95 15; 45; 50; 95 a-TSG
Table 5.5: Molecular weight of tick-tissue antigens (kDa) recognized by different sera of dogs that were either repeated innmunized by using salivary gland (TSG) or midgut (TMG) antigen or multiple infested (INF) by adult females and males of R. sanguineus CHAPTER 6
SYNOPSIS
It is well known that ticks are distributed throughout the world in all countries and different species of ticks are parasitic on many species of animals, either wild or domesticated. Ticks and the diseases they transmit to livestock are a major health problem of animals in almost all developing countries. Either through their direct or indirect effects on animals, ticks causes major economic losses in all pet and livestock industries.
Rhipicephalus sanguineus has been implicated in the transmission of several parasites that cause additional losses for pet owners, including Babesia canis,
B. gibsoni, Ehrlichia canis, and Hepatozoon canis (Ewing 1969), (Seneviratna et al. 1973), (Groves et al. 1975). This tick was also reported as a vector of
Anaplasma marginale (Parker 1982), Coxiella burnetii (Stephen et al. 1980), and Rickettsia conori (Injeyan et al. 1971). Recent reports have shown that R. sanguineus occasionally parasitize humans, indicating a potential role for transmission of zoonotic pathogens as well (Goddard 1989), Carpenter et al.
1990), (Felz, Durden et al. 1996), (Guglielmone et al. 1991).
143 The traditional method to control ticks is application of acaricide that was first introduced in the 1950s and continues today to be almost the only
method of tick control throughout the world. The emergence of tick resistance to acaricides, the accumulation of these chemicals in the environment and their rising costs have resulted in searching into alternative methods of control based on the use of host defense mechanisms. The effect of immunization can be long lasting and without the complication of chemical residues.
Vaccines are environmentally safe and should be very target-species specific.
In addition, arthropod resistance to vaccines through selective adaptation is less likely to occur. Vaccines can also be used to reduce the amount of pesticide necessary for control of an ectoparasite.
A commercial vaccine against B. microplus, a highly adapted one-host tick of cattle, has been developed with the midgut-associated immunogen
Bm 86 (Willadsen and Kemp 1989). This vaccine has been shown to reduce the engorgement weight and egg laying capacity of engorged female ticks.
The success of the B. microplus vaccine indicated that it may be possible to induce immune protection against R. sanguineus in dogs as has been done with guinea pigs. Immune resistance to R. sanguineus may reduce the fecundity and subsequent burdens of the ectoparasite, resulting in less direct damage due to heavy infestation of the host and perhaps curtailing the transmission of tick-borne pathogens.
144 The results of this research Indicated that dogs are good candidate
animals for the study development and practical application of an Ixodid tick vaccine. These animals gradually develop a strong Immunologically mediated tick rejection response. Development of resistance due to successive
Infestations appeared to be less pronounced than direct Immunization, but this
Is not surprising since the dog is the natural host to which R. sanguineus Is highly adapted. The resistance manifested by repeated Infestation affected both feeding and fecundity performances In a manner similar to that of
Immunization with salivary glands. Again, this Is not surprising since the majority of antigens released into the host during feeding are probably secretory products of the tick salivary glands.
The sera of tick-reslstant hosts have been shown to contain antibodies that bind specifically to tick salivary gland and midgut tissues since there was evidence that passive transfer of Immune sera and peritoneal exudate cells from tick-reslstant hosts conferred resistance to tick feeding (Brown et al.
1982). In the resistant host a prominent Increase of antibodies were detected and demonstrated their association with tick tissue Immunogens. Specific antigens from tick salivary glands and midgut have been associated with protective Immunity against different tick species, and these tissues have been proved to Induce resistance to ticks In dogs. These findings suggested that
Immunological vaccination of dogs against R. sanguineus would pose a new alternative for long term control of tick populations and the spread of tick-borne diseases. 145 There are a few important considerations before making an anti-tick vaccine. Vaccination with midgut tissue induces resistance mainly as a reduction in tick fecundity, which has the greatest advantage concerning tick population control. However, this vaccine is unlikely to prevent infection by tick-borne pathogens (Bell et al. 1979) (Wikel et al. 1997). The major role of a putative tick vaccine would be to control tick infestation perse and thus reduce or abolish the need to apply acaricides. The salivary gland vaccine is more effective in preventing feeding and partially effective in depresses fecundity, as well as interference transmitting diseases. Excluding midgut and salivary gland protein, other tick tissues including muscle, synganglion, and reproductive might be considered as candidate antigen if they are capable of inducing resistance against this tick. Apart from prevailing tissues, there were evidences of ectoparasite tissues used as prospective antigen such as thoracic muscles of stable flies, Stomoxys calcitrans, which were used to vaccinate rabbits (De Lello and Boulard 1990; Cross 1993). Both stable flies and tsetse flies {Glossina morsitans), which fed on the immunized rabbits were killed and some survivors showed other effects of vaccination including paralysis of legs, interference in deposition of endocuticle, and reduced post emergence growth (Colwell and Baron 1990) (Baron and Colwell 1991). This evidence has brought the concept of a multivalent vaccine, which consists of components from more than one-tick tissue. This proposed vaccine could prevent ticks from feeding, inhibit tick fecundity, increase tick mortality, and in some cases reduce the transmission of tick-borne pathogens such as 146 Borellia burgdorferi (Wikel et al. 1997). An effective immune response directed
preferentially against different tick stages would be of a supplementary benefit
to the host.
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