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Home , Acetylesterase, Phosphatase

Aust. J. Bioi. Sci., 1976, 29, 487-97

Boophilus microplus: Characterization of Introduced into the Host

A. V. Schleger and D. T. Lincoln Division of Animal Production, CSIRO, Long Pocket Laboratories, Private Bag No.3, Indooroopilly, Qld 4068.

Abstract A number of enzymes, presumably secreted by larvae of B. microplus under natural feeding conditions, have been investigated in the skin of previously unexposed calves 4 h after infestation at the attachment site. Carboxylic activity was demonstrated in the dermis, immediately adjacent to the mouthparts, or in the attachment cone, depending on and reaction pH. The carboxylic ester hydrolase acting on naphthol AS-D (2-acetoxy-3-naphthoic-O-toluidide) at pH 7·1 was characteristically found in the dermis and not in the attachment cone. The use of specific inhibitors showed that this was primarily a B- or with possibly a small portion of C-esterase or . It is postulated that carboxylic ester hydrolase could contribute to the dilation observed in the subepidermal capillaries adjacent to the attachment sites of unexposed animals, through the formation of plasma kinins. Other enzymes demonstrated in the dermis, adjacent to the mouthparts, were triacylglycerol , as an aggregated deposit, and small amounts of aminopeptidase (microsomal) and monophenol monooxygenase. Aminopeptidase (microsomal) was also demonstrated in the attachment cone or adjacent epidermis, according to the substrate used. No activity was found in the host tissue, in association with the attachment site, for either alkaline or acid , or , peroxidase or amine oxidase (flavin-containing), despite the intense histochemical reaction for the latter in the tissues of larvae.

Introduction Tick resistance in cattle, known to be immunologically mediated (Roberts 1968a), is especially manifest during the first 24 h of larval attachment (Roberts 1968b). The saliva secreted by larvae therefore probably contains the antigens against which the host has mounted an immunological response. No information is available on the secretions by tick larvae soon after attachment, studies so far having been restricted to saliva obtained from adult females which have completed the parasitic life cycle and have been stimulated with pilocarpine. Tatchell (1971) obtained a number of esterase and bands in electrophoretic studies on saliva, whilst Geczy et al. (1971) showed that the saliva of adult ticks contains esterase. Tatchell also demonstrated three enzyme types in the haemolymph, namely , and aminopeptidase. If there are enzymes in the saliva of larvae it should be possible to detect them at attachment sites by histochemical techniques. Such enzymes might also be antigens and this could be important in studies of host response and mechanisms of the resistance of cattle to ticks. In the present study enzyme activity at larval attachment sites was investigated on previously unexposed hosts, using histochemical procedures 488 A. V. Schleger and D. T. Lincoln

for carboxylic ester hydrolase, acid and , acetylcholinesterase and cholinesterase, triacylgJyceroJ lipase and three .

Materials and Methods Animals In each investigation a Shorthorn calf, previously unexposed to tick larvae, was used. The unexposed calf was clipped along the loin and a number of chambers, 1· 5 cm in diameter, were fixed to the skin. Approximately 2000 larvae of the Yeerongpilly strain (Stone 1957) were placed in each chamber and confined with nylon gauze. After 4 h the skin under each chamber site was infiltrated with 2 % xylocaine (Astra Chemicals Pty Ltd, North Ryde, N.S.W.) free of adrenaline, and a tissue biopsy was taken with a 1 cm diameter trephine.

Table 1. Commercial and systematic names of substrates, diazonium salts and inhibitors used in histochemical studies

Commercial name Systematic name

Substrates Naphthol AS-D acetate 2-Acetoxy-3-naphthoic-o-toluidide Naphthol AS-TR 3(4-Chloro-2-methyl anilide)-2-naphthyl phosphate

Diazonium salts Fast blue RR 4-Benzoylamino-2,5-dimethoxy aniline Fast blue B 0-Dianisidine Fast red TR 5-Chloro-o-toluidine Fast garnet GBC 4-Amino-3,I-dimethyl azobenzene Hexazonium PR Hexazotized fuchsin (p-rosanilin)

Inhibitors Coroxon 3-Chloro-7-hydroxy-4-methyl coumarin diethylphosphate Iso-OMPA Tetra-isopropylpyrophosphoramide 284C51 1,5-Bis-(4-allyl dimethyl ammonium phenyl) pentan-3-1-diiodide PCMB p-Chloromercuribenzoate

Tissue Processing The biopsies were quenched in a tube of liquid propane surrounded by a jacket of liquid nitrogen, and transferred to a liquid nitrogen cabinet until required. Some tissues were fixed for 4-6 h in 10% neutral formalin at 4°C, and, after washing, held in the cryostat at -20°C. Control data on the enzyme activities of unfed, freshly hatched larvae were obtained by immersing larvae in 20 % gelatin, with 1 % Triton X-I00 added, in an aluminium foil thimble, and the thimble was quenched in liquid propane. The gelatin block was then transferred to the liquid nitrogen cabinet. Tissues were transferred from the liquid nitrogen cabinet to the cryostat in a container cooled with dry ice.

Histochemical Procedure Sections 12.um thick were cut in a cryostat and, for the typing of the esterases, about 20 sections were taken up on each slide. Two slides provided sections through approximately 100 lesions, although different sections might come from the same lesion. After 20 min air drying of the sections at room temperature, the slides were held at 4°C for a minimum time before the reactions were carried out. The commercial and systematic names of substrates, diazonium salts and inhibitors used in this investigation are shown in Table 1. (i) Carboxylic ester hydrolase (EC 3.1.1) The histochemical procedures used for demonstrating esterase activity involved the use of naphthol AS-D acetate substrate (2-acetoxy-3-naphthoic-O-toluidide) at pH 7·1 with Fast blue RR Larval Enzymes of Boophilus microplus 489

coupling agent (Burstone 1962), O-acetyl-5-bromoindoxyl substrate at pH 8· 8 with Fast blue RR coupling agent (Delellis and Fishman 1965; Pearse 1972), and a-naphthyl acetate substrate at pH 7·4 with Fast blue B as coupling agent (Pearse 1972). When naphthol AS-D acetate was used as substrate, the carboxylic ester were classified in this study as A, Band C types as suggested by Pearse (1972), approximately synonymous names being (EC 3.1.1.2), carboxylesterase (EC 3.1.1.1) and acetylesterase (EC 3.1.1.6), respectively. The inhibitors used and their effect on the three types of esterase and cholinesterase are shown in Table 2. The inhibitors and their concentrations are as recommended by Pearse (1972) with the addition of the organophosphate Coroxon which was used at 0·001 M concentration.

Table 2. Inhibitors used in the study of carboxylic ester hydrolases in lesions produced in previously unexposed animals by larvae of B. micropius

Treatment Inhibitors Enzymes affected Enzymes unaffected

1 10 11M eserine Carboxylic ester hydro lases 2 0·001 M CUS04 A-, C-esterase B-esterase 3 0·001 M CUS04 A-, C-esterase, B-esterase + 10 11M eserine cholinesterases 4 O' 001 M Coroxon B-esterase A-, C-esterase 5 0·001 M Coroxon B-, A-esterase C-esterase +0·001 M PCMB

Sections were preincubated for 30 min in 0·2 M tris buffer (pH 7·1) at 37°C, with the appropriate inhibitor, before being transferred to the substrate with the inhibitor again added. Different combinations of inhibitors were used, and different comparisons of inhibitor effects were made, to deduce the contribution of each type of esterase activity. Because of the varying number of lesions appearing in the same number of sections, inactive lesions were expressed as a percentage of the total. The rationale of the esterase typing is shown in Table 3.

Table 3. Carboxylic ester hydrolase activity deduced from the increase in inactive lesions through the use of specific inhibitors according to Table 2 Inactive lesions showed no enzyme activity

Difference between treatments (Table 2) Carboxylic ester hydrolase activity

1-(Treatment 2+treatment 3)-treatment 1 A- and C-esterase Treatment 5-treatment 4 A-esterase Treatment 5-treatment 1 B- and A-esterase Treatment 4-treatment 1 B-esterase

Lesions were assessed as inactive (-), i.e. showing no enzyme activity, or as having three degrees of intensity of histochemical reaction: +, + + and + + +. It was assumed that the loss of activity through the selective inhibition of one or more esterase components resulted in an increase in the number of inactive lesions, and the percentage of inactive lesions was used as an index of inhibitor effects. For that reason, intensity of reaction in the positive lesions was not considered in the data analysis.

(ii) Acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) The activity of acetylcholinesterase and cholinesterase was Idemonstrated by the method of Koelle and Friedenwald (1949), using acetylthiocholine iodide and butyrylthiocholine iodide as substrates. The inhibitors used in the distinction between acetylcholinesterase and cholinesterase activity were as recommended by Pearse (1972). 490 A. V. Schleger and D. T. Lincoln

(iii) Triacylglycerollipase (EC 3.1.1.3) The method of Gomori (1952), using Tween 80 or Polysorbate 80 (a complex mixture of polyoxyethylene ethers of mixed partial oleic of sorbitol anhydrides) at pH 7·3 as substrate, was used to demonstrate lipase. (iv) Aminopeptidase (microsomal) (EC 3.4.11.2) The demonstration of aminopeptidase (microsomal) was based on the method of Ackerman (1960). Two substrates were used~alanyl-;9-naphthylamide and l-leucyl-4-methoxy-;9-naphthylamide~at pH 6· 7 and two coupling agents~Fast garnet GBC and Fast blue B (Table 1).

(v) Acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) Acid phosphatase activity was demonstrated by the method of Barka and Anderson (1962), using naphthol AS-TR phosphate at pH 5·0 as substrate and hexazonium PR as the coupling agent. Alkaline phosphatase was demonstrated using the same substrate at pH 8· 0 and Fast red TR as the diazonium salt (Burstone 1962). (vi) Oxidoreductases The methods for monophenol monooxygenase (EC 1.14.18.1) and amine oxidase (flavin-containing) (EC 1.4.3.4) were taken from Pearse (1968) and were based on the methods of Becker et al. (1935) and Glenner et al. (1957). Peroxidase (EC 1.11.1.7) was demonstrated by the method of Graham et al. (1965).

Results Carboxylic Ester Hydrolases The distribution of carboxylic ester hydrolase activity differed according to the substrate used. (i) Naphthol AS-D acetate When naphthol AS-D acetate was used as substrate, the carboxylic ester hydrolase activity was almost completely confined to the dermis, immediately subjacent to the attachment site (Fig. 1). No activity was detected in the attachment cone. In some biopsies the epidermal cells showed a reaction for some distance from the feeding cone. This could indicate uptake of the enzyme by the epidermal cells. Apart from the larval attachment sites, there was no hydrolase activity, as demon­ strated by naphthol AS-D acetate, in the formalin-fixed host tissue. Slight activity was observed in the secretory cells of the sebaceous glands in the sections from the quenched biopsies. (ii) O-Acetyl-5-bromoindoxyl The hydrolase activity in the larval lesion when O-acetyl-5-bromoindoxyl was used as substrate was mostly restricted to the attachment cone (Fig. 2). Only a small proportion of lesions contained significant activity in the subjacent dermis. No other activity was seen in the epidermis. In the epidermal appendages enzyme activity was observed in the outer root sheath of hair follicles in the catagen phase, especially in the region of the hair bulge, and in the orifice of sebaceous glands. (iii) IX-Naphthyl acetate The hydrolase activity demonstrated with IX-naphthyl acetate as substrate was more widely distributed at the attachment site (Fig. 3) and was found in both the cone and subjacent dermis. There was a reaction throughout the epidermis, but the intensity was much less than that at the attachment site and was easily distinguishable. The activity was more widely distributed in the host tissue in this reaction than it was Larval Enzymes of Boophilus micropius 491

in the reaction using O-acetyl-5-bromoindoxyl. In addition to the intense reaction of the sebaceous gland orifice, the secretory cells had a faint reaction. The larger dermal blood vessels, especially arteriovenous anastamoses, showed a positive reaction.

Figs 1-3. Distribution of carboxylic ester hydrolase activity in the 4-h larval lesion for naphthol AS-D acetate at pH 7·1 with Fast blue RR as coupling agent (Fig. 1), for O-acetyl-5-bromoindoxyl at pH 8· 8 with Fast blue RR as coupling agent (Fig. 2), and for (X-naphthyl acetate at pH 7· 4 with Fast blue B as coupling agent (Fig. 3). In Fig. 1 the activity is confined to the adjacent dermis, whilst in Fig. 2 the activity is largely confined to the attachment cone although limited activity may be observed in the adjacent dermis. The enzyme activity shown in Fig. 3 is virtually a summation of the distributions shown in Figs 1 and 2. Fig. 4. Triacylglycerollipase activity, using Tween 80 at pH 7· 3. The aggregated state of the enzyme reaction is apparent. 492 A. V. Schleger and D. T. Lincoln

Type of Carboxylic Ester Hydrolase in Naphthol AS-D Acetate Reaction The percentage of inactive lesions is set out in Table 4 for the naphthol AS-D acetate reaction using various inhibitors. The results for the two biopsies are listed separately. The main inhibition of enzyme activity was produced by the organophosphate Cor'oxon at 0·00 I M concentration (treatment 4). The proportion of inactive lesions increased to 55 % in biopsy 1 and to 74 % in biopsy 2. This indicated that B-esterase was a major component of the enzyme mixture.

Table 4. Total number of lesions per 40 sections from each of two biopsies, and percentage of inactive lesions produced by each inhibitor treatment, in determining type of carboxylic ester hydrolase involved in the naphthol AS-D acetate reaction Treatments are as in Table 2

Treatment Biopsy 1 Biopsy 2 Total Percentage Total Percentage lesions inactive lesions inactive

No preincubation 85 5 177 16 Preincubation only 71 7 228 33 1 67 21 134 17 2 103 27 109 34 3 105 27 147 37 4 71 55 148 74 5 95 60 74 58

There is evidence (Table 4) that the experimental error may be as high as 16 %. Preincubation produced no change in the percentage of inactive lesions in biopsy 1, but an increase of 17 % in biopsy 2. On the other hand, 10 j,tM eserine (treatment 1) produced no increase in biopsy 2, which was expected in view of the absence of cholinesterase in the lesion, but an increase of 16 % in biopsy 1. Likewise, the com­ bination of 0·001 M PCMB with 0·001 M Coroxon (treatment 5) produced no significant increase above 0·001 M Coroxon alone (treatment 4) in the percentage of inactive lesions in biopsy I, but a decrease of 16 % in biopsy 2, which is untenable. The increase in the percentage of inactive lesions produced by 0·001 M CuS04 (treatment 2) was within the limits of experimental error and so, on this basis, C­ esterase could not be assumed present. On the other hand, there was some strong residual activity after treatment with a combination of Coroxon and PCMB (treatment 5). This suggests the presence of C-esterase which is activated by PCMB. The estimated activities of A-, B- and C-esterase are indicated in Table 5. The scheme is the same as in Table 3, except that mean percentage values were used where treatments were carried out with and without 10 j,tM eserine. For example, the basic percentage of inactive lesions (Table 5) was derived from the mean of 'preincubation only' and treatment 1 (Table 4). Since 10 j,tM eserine has no effect on non-specific carboxylic ester hydrolases, it should not alter the percentage of inactive lesions any more than preincubation only. The relative proportion of the three esterase types in each biopsy may be deduced, but the values for A-esterase and possibly C-esterase are of doubtful significance in view of the error involved. Larval Enzymes of Boophilus microplus 493

Acetylcholinesterase and Cholinesterase A strongly positive reaction was obtained in the larval brain, using acetylthio­ choline iodide as substrate, whilst a faintly positive reaction was obtained with butyrylthiocholine iodide. In some lesions a positive reaction for acetylcholinesterase activity was demonstrated in the intraepidermal portion of the attachment cone, but no activity was observed in the adjacent dermis.

Table 5. Type of carboxylic ester hydrolase activity deduced from the change in inactive lesions brought about by the use of specific inhibitors according to Table 3

Equivalent percentage of inactive lesions Biopsy 1 Biopsy 2

Basic inactive lesions 14 25 A- and C-esterase 13 11 A-esterase 5 o B- and A-esterase 46 33 C-esterase 8 11 B-esterase 41 33

The presence in larval tissue of an enzyme reacting with butyrylthiocholine substrate prompted an investigation of this enzyme in larvae devoid of any contact with host tissue. Table 6lists the inhibitors used in this study. The selective inhibitors for , I {1M iso-OMPA and 25 {1M sodium , had no signi­ ficant effect on the reaction product even when used at double the recommended strength. On the other hand, 50 {1M 284C51 (Wellcome Reagents, Beckenham, U.K.), which produces complete inhibition of acetylcholinesterase, almost completely inhibited the enzyme as it did when acetylthiocholine iodide was used as substrate.

Table 6. Inhibitors used in the investigation of cholinesterase activity in unattached larvae, and the degree of inhibition produced using acetyithiocholine iodide and butyrylthiocholine iodide as substrates

Substrate Eserine 284C51 NaF Iso-OMPA Iso-OMPA (1·2 /.lM) (10/.lM) (50/.lM) (25·50/.lM) (1·2/.lM) +284C51 (50/.lM)

Butyrylthiocholine Complete Almost Little Little Almost iodide complete effect effect complete Acetylthiocholine Complete Almost _A _A _A iodide complete

A Inhibitors not used on substrate.

Triacylglycerol Lipase The lipase reaction, using Tween 80 as substrate, gave a particulate product in the vicinity of the lesion (Fig. 4). No other reaction was given by the host tissue.

Aminopeptidase (Microsomal) An intense red reaction for aminopeptidase (microsomal) was given by the attach­ ment cone, using 1-leucyl-4-methoxy-~-naphthylamide as substrate and Fast garnet GBC as coupling agent (Fig. 5). When alanyl-~-naphthylamide was used as substrate, and Fast blue B as the coupling agent, an orange reaction product appeared in the 494 A. V. Schleger and D. T. Lincoln

epidermis adjacent to the cone (Fig. 6). In each case a small amount of reaction product appeared in the subjacent dermis. The enzyme activity of the larvae was very intense. In the host tissue a strong reaction was given by the degenerating cells of the dermal papilla, in the resting phase of hair growth, and a moderate reaction by the secretory cells of the sweat glands.

Figs 5 and 6. Distribution of aminopeptidase (microsomal) activity for alanyl-p-naphthylamide at pH 6·7 with Fast garnet GBC as coupling agent (Fig. 5), and for l-leucyl-4-methoxy-p­ naphthylamide at pH 6' 7 with Fast blue B as coupling agent (Fig. 6). The intense red reaction product in Fig. 5 was confined to the attachment cone, whilst a limited, faint reaction product (arrows) was found in the adjacent dermis. The light brown reaction product in Fig. 6 occurred in the epidermis although limited activity was again observed in the adjacent dermis.

Acid Phosphatase and Alkaline Phosphatase There was no increase in phosphatase activity near the attachment site although there was some evidence that the acid phosphatase activity of macrophages, when present in the subjacent dermis, was increased.

Oxidoreductases Three oxidoreductases were investigated-monophenol monooxygenase, amine oxidase (flavin-containing) and peroxidase. There was a slight but consistent oxidation of DOPA in the host tissue immediately subjacent to the mouthparts. Peroxidase activity was not detected at this location so that this reaction was not peroxidase mediated. An intense amine oxidase (flavin-containing) reaction was given by the larval tissue but no enzyme activity was observed at the attachment site.

Discussion A number of enzymes, presumed to be secreted by larvae under natural feeding conditions, have been demonstrated in the skin of previously unexposed calves. The presence of enzymes at the attachment site, but absent from the remainder of the epidermis or subepidermis, indicated that they were derived from the parasite. Larval Enzymes of Boophilus microplus 4')5

Indeed, in some cases the enzyme activity associated with the larval mouthparts was the only activity detected in the host tissue. Induction of enzyme activity by host substrate is most unlikely since the larva itself contained high enzyme activity, the activity in some cases was confined to the attachment cone, and activity in hosts with previous tick experience appeared less. The carboxylic ester hydrolase demonstrated in the dermis immediately subjacent to the larval mouthparts with the substrate naphthol AS-D acetate was shown, through the action of specific inhibitors, to be primarily a B-esterase. There was some indication of the presence of C-esterase, but no evidence for A-esterase. The non-specific esterase found in the perienteric fluid of Ascaris suum appears to be a B-esterase (Tan and Zam 1973). Whilst B-esterase is the major enzyme found in strigeoid trematodes, both A- and C-esterases have also been detected (Tieszen et al. 1974). The non-specific esterase injected into the host tissue by the larvae could contribute to the dilation in the subepidermal capillaries described in unexposed animals (Tatchell and Moorhouse 1968; Tatchell and Binnington 1973). Esterases induce the formation of plasma kinins, which are potent vascular permeability­ inducing agents (Movat 1971). Neither acetylcholinesterase nor cholinesterase was detected in the dermis at the attachment site. Acetylcholinesterase, however, was found in the intra-epidermal portion of the feeding cone. Enzyme activity was shown in the larval brain using acetylthiocholine and, to a lesser extent, butyrylthiocholine substrates. Acetyl­ cholinesterase has been demonstrated in the brain of the cattle tick (Stone 1968). There is little evidence for the presence of cholinesterase in arthropods (Treherne 1966), and so a significant reaction product for this enyzme, especially when emphasized by silver nitrate treatment as in the Henderson (1967) modification, called for further study. The presence of enzyme activity when butyrylthiocholine substrate was used suggested the presence of cholinesterase as well as acetylcholinesterase (Pearse 1972). The apparent cholinesterase activity was not inhibited by iso-OMPA or sodium fluoride, selective inhibitors for cholinesterase, even at twice the recommended concentrations (211M and 50 11M respectively). On the other hand, it was inhibited by 50 11M 284C51, which specifically inhibits acetylcholinesterase. This suggests limited activity by acetylcholinesterase with butyrylthiocholine iodide, and such activity has been demonstrated with extracts of tick larvae (J. Nolan, personal communication). The presence of in the dermis subjacent to the mouthparts is of interest, since Dailey and Hunter (1974) presented evidence that the type of immune response is not so much a function of the antigenic determinant but of the entire immunogen, lipid-rich strongly hydrophobic carriers being necessary for a delayed-type hypersensitivity reaction. The breakdown of any lipoprotein moiety of the larval saliva might ensure an immediate rather than a delayed reaction in the host. The possibility that the lipase contains a significant proportion of is currently being investigated. Phospholipase A, derived from hymenopteran venoms, has a number of pharmacological and biochemical properties (Habermann 1972). In addition to an increase of capillary permeability and tissue damage, it can produce 'indirect' haemo1ysis, thromboplastin inactivation and interruption of electron transport and oxidative . Moderate doses of phospholipase A depress the level of plasma phospholipids in rabbits (Habermann 1972). The same 496 A. V. Schleger and D. T. Lincoln

effect might be produced by tick infestation in cattle. O'Kelly and Seifert (1969) showed that blood phospholipid decreased during tick infestation when the level of nutrition was below maintenance. Phospholipase A is one of the two enzyme com­ ponents of bee venom which are antigenic (Habermann 1972). A major difference between the enzymes associated with the attachment of larvae and those detected by Tatchell (1971) in the saliva of adult ticks seems to be the relative proportions of aminopeptidase and acid phosphatase. Aminopeptidase (microsomal) appeared in high concentration in the larvae and there was high activity in the attachment cone. A lower degree of activity occurs in the adjacent epidermis and dermis. On the other hand, aminopeptidase was a minor constituent of adult tick saliva. Whilst acid phosphatase was second in importance to esterase in the adult saliva, it was not detected in larval lesions. A small amount of monophenol monooxygenase activity was consistently found below the larval mouthparts. No peroxidase activity was detected at the attachment site, and so this activity was not peroxidase-mediated (Okun et aZ. 1970). The high concentration of monoamine oxidase reported for B. micropZus by Atkinson et af. (1974) was confirmed in these studies. Although there is considerable knowledge of the composition, physiological effects and antigenicity of hymenopteran venoms (Habermann 1971), very little is known of the secretion of other arthropods. The histochemical procedures described can be used to investigate the nature of the secretions of parasites at any stage of the parasitic life cycle. It is anticipated that the enzymes described are also antigens, which will facilitate studies of the immune response of the host and its relationship to the mechanism determining the degree of resistance of cattle to the tick B. microp/us.

Acknowledgments The authors wish to acknowledge the photographic assistance of Messrs K. Rode-Bramanis and D. S. Fiske. This work was supported in part by the Australian Meat Research Committee.

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Manuscript received 11 November 1975