Immunology 1976 30 479
Acquired resistance to ticks It. EFFECTS OF CYCLOPHOSPHAMIDE ON RESISTANCE
S. K. WIKEL & J. R. ALLEN Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Received 1 September 1975; acceptedfor publication 26 September 1975
Summary. Guinea-pigs developed resistance to Dermacentor andersoni larvae after one infestation. Cyclophosphamide administered in one dose (300 mg/kg) 48 h prior to an initial infestation with larvae blocked the acquisition of resistance. When cyclophosphamide was given in a similar regimen to guinea-pigs which had already acquired resistance, the expression of resistance was partially blocked. It was proposed that the blockage of the acquisition of resistance further confirmed the immunological nature of tick resistance. Partial blockage of the expression of resistance by cyclophosphamide substantiated the presence of a humoral component to the resistance mechanism. The presence of a cellmediated component was previously well established.
INTRODUCTION
Guinea-pigs acquired resistance to the ixodid tick, Dermacentor andersoni, after one infestation (Allen, 1973). Resistance was characterized by resistant guinea-pigs allowing fewer larvae to engorge (5-25 per cent) than a non-resistant host (70-95 per cent); larvae engorging on a resistant host took a significantly smaller bloodmeal than larvae feeding on a non-resistant host (Wikel and Allen, 1976). Resistant animals displayed a histological response character-
ized by epidermal hyperplasia, intra-epidermal vesicles and an intense infiltration of the epidermis and vesicles with basophils (Allen, 1973). An immunological basis for the acquisition of resistance has been proposed by several authors working with ixodid ticks infesting laboratory animals (Trager, 1939; Garin and Grabarev, 1972; Boese, 1974; Bagnall and Doube, 1975). Previous communications from this laboratory established that acquired resistance to Dermacentor andersoni larvae by guinea-pigs has an immunological basis. Allen (1973) showed that the immunosuppressant Methotrexate blocked the acquisition of resistance to ticks. Wikel and Allen (1976) reported that resistance to ticks was passively transferred with viable lymph node cells, but not serum, from resistant guinea-pigs. Cyclophosphamide has been used extensively to investigate immune responses in guinea-pigs (Turk, Parker and Poulter, 1972; Turk and Poulter, 1972; Neta and Salvin, 1974). This report concerns attempts to regulate the acquisition and expression of resistance to ticks with cyclophosphamide. MATERIALS AND METHODS Animals Randomly bred guinea-pigs used in these experiments were obtained from the Animal Resources
Correspondence: Dr S. K. Wikel, Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO.
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S. K. Wikel & J. R. Allen
Centre, University of Saskatchewan. They were of either sex and weighed between 450 and 650 g.
Control animals
One intraperitoneal dose of cyclophosphamide (300 mg/kg)
(Similar regimen to experimental animals with exception of
Initial infestation with 100 larvae 5 days Removal of larvae and recording of number engorged and unengorged |7 days
I48h Parasites Dermacentor andersoni larvae were supplied from the pathogen free colony of the Rocky Mountain Laboratory (NIAID, Hamilton, Montana). Larvae were maintained at 150 in cotton-plugged vials in a desiccator over a saturated solution of potassium nitrate. All larvae used during any particular experiment were derived from one female tick; thus, larvae used during each experiment would have a uniform feeding capability. In each experiment, non-resistant and resistant control guinea-pigs were included (alongside test animals) to provide a comparison of the feeding activities of the larvae on these and experimental hosts. During infestation larvae were contained in plastic capsules, approximately 15 mm in diameter, fitted over the ear and held in place by adhesive tape. This restricted the larvae to the ear and prevented grooming by the animal. At the end of an infestation period the number of engorged and unengorged larvae was determined by examination using a stereoscopic microscope.
Cyclophosphamide Cyclophosphamide (Procytox, Frank W. Horner, Limited, Montreal, Canada) was dissolved in 0 85 per cent sodium chloride and injected in one intraperitoneal dose of 300 mg/kg body weight.
cyclophosphamide)
Second infestation with 100 larvae |5 days
Removal of larvae and recording of number engorged and unengorged |7 days Third infestation with 100 larvae 5 days
Removal of larvae and recording of number engorged and unengorged
Fig. 1. Design of experiment to block acquisition of resistance by use of cyclophosphamide.
Initial infestation with 100 larvae
5 days Removal of larvae and recording of number engorged and unengorged 5 days
One intraperiloneal dose of cyclophosphamide (300 mg/kg) 48 h
Experimental design In experiments designed to block the acquisition of resistance, cyclophosphamide was administered 48 h before the initiation of a primary infestation with 100 larvae. After 5 days, the number of larvae engorging was determined. Seven days after the termination of an initial infestation a second 5-day infestation with 100 larvae was begun. The number of larvae engorging was determined. If the acquisition of resistance was blocked, the number of larvae engorging during the first and second infestations should be nearly similar. A third 5-day infestation with 100 larvae was initiated 7 days after the termination of the second exposure. Experiments designed to block the expression of resistance followed a different scheme. Guinea-pigs were exposed to an initial infestation with 100 larvae for 5 days in order to acquire resistance. The number of larvae engorging was determined. These animals
Infestation with 100 larvae
5 days Record number of larvae engorged; weigh larvae after fixation and drying
Control 48 h
Infestation with 100 larvae 5 days
Record number of larvae engorged; weigh larvae after fixation and drying
Fig. 2. Design of experiment to block expression of resistance by use of cyclophosphamide.
were then divided into two groups. The first group after 5 days was given one dose of cyclophosphamide and 48 h later received an infestation with 100 larvae. The second group was treated in a similar fashion; except these animals did not receive cyclophosphamide. After 5 days the number of larvae engorging in each group was determined. Figs 1 and 2 give an overview of the experimental design. A total of sixty animals were used in these
experiments.
Acquired resistance to ticks. II.
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Table 1. Effects of cyclophosphamide on acquisition of resistance. Percentages of D. andersoni larvae engorging on guinea-pigs (mean+ s.e.)
Guinea-pig groups: Treatment:
A Cyclophosphamide prior to first infestation
B
C
D
Saline prior to first infestation
Nil
Nil
94_7+4-0 84-4+ 14-2 23-4+4-4
770+ 3-1 26-8+0-0 25-6+00
95-7+0-1 9 5+35
80-2+0-0
Infestation 1 Infestation 2 Infestation 3
Weight of larvae Larvae were collected from each host in the experiments designed to measure the blocking of the expression of resistance and placed into 10 per cent formalin for fixation. Larvae were removed from fixative, blotted, placed on filter paper and allowed to dry for 6 h at 600. Larvae from each host were weighed and the mean weight determined. Weights were determined to see if the visual assessment of engorgement corresponded with the bloodmeal taken by each group of larvae. Histology of lymph nodes Cervical lymph nodes were removed from cyclophosphamide recipients and control animals, fixed in 2 per cent glutaraldehyde-5 per cent formaldehyde in 0-05 M phosphate buffer. Nodes were dehydrated in graded ethanols and embedded in paraffin via chloroform. Sections were stained with Giemsa. RESULTS Effects of cyclophosphamide on acquisition of resistance
Cyclophosphamide administered before a primary infestation with D. andersoni larvae effectively blocked the acquisition of resistance to a subsequent larval infestation (Table 1). Group A animals received cyclophosphamide prior to infestation one, while group B animals acting as normal controls received saline prior to their first infestation. The number of larvae engorging during infestation one in both groups A and B was within the range expected for a primary infestation. However, significantly more larvae engorged on group A animals than on group B animals (P