Protective efficacy of doramectin and ivermectin against Cochliomyia hominivorax

veterinary parasitology ELSEVIER

Veterinary Parasitology72 (1997) 101-109

Protective efficacy of doramectin and ivermectin against Cochliomyia hominivorax G.E. Moya-Borja a, R.A. Muniz u,1, O. Umehara c, L.C.B. Goncalves c, D.S.F. Silva a, M.E. McKenzie b,, a Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil b Animal Health Group, Pfizer, 235 East 42nd Street, New York, NY 10017, USA c Pfizer, Sao Paulo, Brazil

Abstract Two studies were conducted in Brazil using induced infestations of the New World screwworm, Cochliomyia hominivorax, to investigate: a) the comparative prophylactic efficacy of doramectin and ivermectin at their recommended use levels (200 /xg kg - l SC), and b) the duration of protection of a single injection of doramectin. In the comparative efficacy study, two groups of six animals each were treated with ivermectin or doramectin. Two hours after treatment, four incisions were made. Each incision was infested with 30 first instar C. hominioorax larvae and their status evaluated daily for 7 days post-treatment (p.t.). Doramectin treatment was 100% effective in prevention of C. hominivorax infestations whereas ivermectin efficacy was incomplete. First instar larvae were eliminated in dorarnectin-treated calves by 48 h p.t., while in the ivermectin group, C. hominivorax developed in over 29% of the incisions. Healing began in wounds of doramectin-treated animals at 24 h p.t. and was in progress in 100% of all wounds at 2 days p.t., while 50% of ivermectin-treated calves showed incisions with active lesions. In the duration of protection study, 24 calves were allocated to six groups (T1-T6) of four animals each. Three groups (T1, T3 and T5) were treated with saline and three groups (T2, T4 and T6) with doramectin. Animals were infested as described previously according to the following schedule: T1 and T2 at day 14, T3 and T4 at day 21, and T5 and T6 at day 28 p.t. Incisions were evaluated dally for 8 days post-infestation. Screwworm infestations and viable third-instar larvae developed of all incisions of saline-treated calves, while doramectin was 100% effective preventing development of C. hominivorax for 21 days p.t. and showed partial activity at 28 days p.t. © 1997 Elsevier Science B.V. Keywords: Doramectin;Ivermectin;Cochliomyia hominivorax; Cattle-arthropoda;Control methods-arthropoda

* Corresponding author. i Deceased. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PH S0304-4017(97)00082-4

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1. Introduction

The protective efficacy of doramectin (Dectomax TM, Pfizer) against the New World screwworm, Cochliomyia hominivorax, had been previously described against induced (Moya-Borja et al., 1993a) and naturally occurring myiasis (Muniz et al., 1995a,b). Moya-Borja et al. (1993a), demonstrated a period of protection of 14 days post-treatment (p.t.), in a study in which the end-point of the persistent efficacy of doramectin was not determined. The period over which cattle are protected against screwworm infestation after treatment is important because it allows the integration of prophylactic medication with routine husbandry practices, such as calving, branding, castration and dehorning. Initial reports described lack of efficacy of ivermectin (Ivomec TM, Merck, Rahway, N.J., USA) against C. hominivorax infestations (Tregoning, 1993; Benz et al., 1989). More recently, Benitez-Usher et al. (1993) reported significantly ( P < 0.01) lower incidence of navel and scrotal myiasis under conditions of field exposure in ivermectintreated animals compared to untreated controls. However, 100% protection of treated animals was not achieved in those experiments. The objectives of the studies reported here were: a) to compare the protective efficacy of doramectin and ivermectin under the same conditions of induced challenge, and b) to determine the duration of the protective activity of doramectin.

2. Materials and methods

2.1. Study site and animals Two studies were conducted at the School of Veterinary Medicine of the Universidade Federal Rural do Rio de Janeiro, Brazil. Animals were kept in individual pens inside fly-proof facilities. Experimental animals were male, cross-breed Holstein by Zebu calves, from 6 to 11 months of age, weighing from 74 to 177 kg with no previous history of myiasis. Calves were selected from a single herd on the basis of uniformity of weight. During adaptation periods (10 to 25 days), animals were dewormed with levamisole (7.5 mg kg -j ) and sprayed with triatox at a concentration of 0.025% to remove ticks. Two to seven days before infestation calves were washed with neutral soap and water to remove any residual acaricide.

2.2. Treatment All calves were treated by subcutaneous injection in the lateral midline of the neck. In study 1, one group received doramectin at 200 /zg kg-1 and the other ivermectin at 200 /_tg kg t of live weight (1 ml 50 kg ~). In study 2, calves in the control group received saline solution at a dose of 1 ml 50 kg ~ of body weight, and the medicated group was treated with doramectin at 200 /xg kg ~ of body weight (1 ml 50 kg-1). In both studies, animals were observed for 6 h post-treatment (p.t.) for signs of adverse reactions. At the end of the experiment calves with active screwworm lesions were individually treated until all wounds were completely healed.

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2.3. Experimental design 2.3.1. Study 1: comparative prophylactic efficacy of doramectin and ivermectin On the day of treatment (Day 0), calves were ranked by weight and randomly allocated in pairs to two treatment groups of six animals each. Two hours after treatment animals were given a local anesthetic and four incisions (two on each side) were made. Each incision was infested with 30 recently hatched first instar C. hominivorax larvae, for a total of 120 larvae per animal, following the procedure described by Moya-Borja et al. (1993a). From Day 0 to Day 7 p.t., calves were examined daily and the healing process of the incisions evaluated and scored using the following criteria: 0, if incision was healed and covered by a scab; 1, healing was in progress and no inflammatory changes or larvae were present; 2, incision was infested and showed bleeding and later presence of exudate and larvae. Third instar larvae that completed development and exited wounds were collected and incubated to verify viability. 2.3.2. Study 2: duration of protection On the day of treatment (Day 0), 24 calves were weighed and individually ranked in descending order on the basis of body weight. The first six calves were randomly allocated to six treatment groups, and the procedure was repeated until all 24 animals were allocated to the six treatments. Calves of groups T1, T3 and T5 received saline and calves of groups T2, T4 and T6 received doramectin. At 14, 21 and 28 days p.t., animals of each paired group were infested as previously described. Calves were examined daily for 8 days p.t. for presence of myiasis caused by C. hominivorax and the healing process of each incision was evaluated and scored as previously described. Third instar larvae that completed development and exited the wounds were collected and incubated to verify their viability. 2.4. Parasitological techniques The C. hominivorax isolates were obtained using a modified screwworm, wind-oriented trap baited with fresh liver (Broce et al., 1977). First instar larvae used for induced infestations were hatched from eggs in the laboratory and were used for infestation within 6 h. Third instar larvae were incubated at 25°C and 70% relative humidity to verify their viability and development to adult flies.

2.5. Statistical analysis The percentage reduction in myiasis was calculated dally by comparing the number of infested wounds (myiases) in treated animals at Day 0 with the number of myiases at Days 1, 2, 3, 4, 5, 6, 7 and 8 post infestation, using the following formula: Percent reduction = (Mean no. of myiases in treated group at Day 0 - - M e a n no. of myiases in treated group at Days 1-8 or 1 - 7 ) / ( M e a n no. of myiases in treated group at Day 0) × 100.

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Table 1 Mean number of infested wounds (myiasis) in calves treated with doramectin or ivermectin Day on test

0 1 2 3 4 5 6 7

lvermectin-treated calves

Doramectin-treated calves

Mean no. of myiases per animal

Mean no. of larvae per animal *

Mean no. of myiases per animal

Mean no. of larvae per animal *

4.0 1.7 1.2 t.2 1.0 1.0 0.8 0.3

0 0 0 0 0 2.5 ~ 3.3 a 8.2"

4.0 0.17 0 0 0 0 0 0

0 0 0 0 0 0b 0b 0b

* Mean number of larvae is the total number of third instar larvae that completed their cycle and abandoned the wounds divided by the total number of animals (6). a'bAcross treatment values with different superscripts are significantly different ( P < 0.05).

In study 2, the number of myiases and the number of larvae recovered from calves of the control group are reported to verify the progression of the infestation in the absence of treatment. In both studies, the geometric means of all third instar larvae, pupae and adult flies of each treatment were compared using analysis of variance on the natural log, (count + 1). The level of significance was P < 0.05. Myiasis and wound healing were analyzed using Categorical Linear Modeling. (SAS Institute, Cary, NC). 3. Results

3.1. Comparative prophylactic efficacy Results of this study are presented in Table 1 and Fig. 1. Doramectin was 100% effective preventing development of C. hominicorax infestations whereas the efficacy of

Ivermectin

Doramectin

24

24 20

GO O 16 Z

16

U. 12

12

n" 8 LU m

8

m

o

4

Z o

0

1

2

3

4

5

6

7

0

0

1

2

3

4

DAYS POST-INFESTATION Fig. 1. Progression of incision healing in comparative prophylactic studies.

5

6

7

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i v e r m e c t i n w a s i n c o m p l e t e . T h e r e w e r e n o a c t i v e l e s i o n s in d o r a m e c t i n - t r e a t e d a n i m a l s at 4 8 h p.t., w h i l e in t h e i v e r m e c t i n - t r e a t e d g r o u p , C. h o m i n i v o r a x l a r v a e d e v e l o p e d f r o m first to t h i r d instars. H e a l i n g b e g a n in t h e d o r a m e c t i n g r o u p at 24 h p.t. a n d w a s in p r o g r e s s in 1 0 0 % o f the w o u n d s at 2 d a y s p.t., w h i l e o n t h e s a m e d a y 2 9 % o f the i n c i s i o n s in i v e r m e c t i n - t r e a t e d c a l v e s s h o w e d e v i d e n c e o f b l e e d i n g , s e r o u s o r p u r u l e n t e x u d a t e , a n d l a t e r t h e p r e s e n c e o f C. h o m i n i v o r a x larvae. B y t h e e n d o f the o b s e r v a t i o n p e r i o d ( d a y 7 p.t.), 8 3 % o f t h e i n c i s i o n s in d o r a m e c t i n - t r e a t e d c a l v e s h a d h e a l e d a n d in the r e m a i n i n g 17%, h e a l i n g w a s in p r o g r e s s . I n t h e i v e r m e c t i n g r o u p , t h r e e c a l v e s s h o w e d f r o m o n e to t h r e e i n c i s i o n s w i t h a c t i v e l e s i o n s o f C. h o m i n i v o r a x a n d in t h o s e

Table 2 Persistent efficacy of doramectin against screwworm Days post-infestation

Saline-treated calves Mean no. of Mean no. of myiases per larvae per animala animalb

Doramectin-neated calves Mean no. of Mean no. of myiases per larvae per animala animalb

Percentage reduction in myiases

Infestation on Day 14 post-treatment (4 calves per group) 0 4 0

4

0

0

1

4

0

2

0

50

2 3 4 5 6 7 8

4 4 4 3 1 0 0

0 0 69 19 18 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

100 100 100 100 100 -

0 100 100 100 100 100 100 -

Infestation on Day 21 post-treatment (4 calves per group) 0 4 0

4

0

1

4

0

0

2 3 4 5 6 7 8

4 4 4 3.5 0.25 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

Infestation on Day 28 post-treatment (4 calves per group) 0 4 0

4

0

0

1

4

1.75

0

56

2 3 4 5 6 7 8

4 4 4 3.5 0.25 0 0

1.75 1.75 1.75 1.5 0.25 0 0

0 0 2.15 2.75 0.5 0 0

56 56 56 57 0 -

0

0 0 12 35 0.25 0 0

0

0 0 52 27 0.25 0 0

alnfective dose: 30 first instar larvae per incision (four incision per animal). bThird instar larvae collected.

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Challenge: Day 14 Post-Treatment Controls

4~

Doramectin 16

,

.

.

.

.

14 12 10

!

0

1

2

3

4

5

6

Z

7

8

,0

0

t

2

3

4

5

6

7

8

6

7

8

DAYS POST-INFESTATION

Challenge: Day 21 Post-Treatment

0

Controls

Doramectin

0

Z m

u_

0 5

UJ CO

DAYS POST-INFESTATION

Challenge: Day 28 Post-Treatment Controls

Z

Doramectin

m

14 , , 12

10 8 6

L~N

4

N

2 0

0

1

2

3

4

5

6

7 8 DAYS POST-INFESTATION

Incision Progression:

8

2 = Infested 1=

Healing in Progress ~

0 = Healed

~-~

Fig. 2. Progression of incision healing in persistent efficacy studies.

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animals wound healing began only after C. hominivorax larvae had completed development and exited the wounds to initiate pupation. A total of 84 viable third instar larvae were recovered from wounds of the ivermectin group, while none were obtained from doramectin-treated calves. All larvae that exited the wounds developed into adult flies. None of the ivermectin or doramectin treated animals exhibited any clinical signs of adverse reaction to medication. 3.2. Duration of protection

Results of this study are presented in Table 2 and Fig. 2. Doramectin injections were 100% effective preventing development of C. hominivorax infestations at 14 (T2) and 21 days (T4) p.t. In these two doramectin-treated groups, there were no active lesions by 24 h post-infestation (p.i.) and healing was completed by 4 and 6 days p.i., respectively. In calves of the corresponding control groups, infestations were active until day 6 p.i. when C. hominivorax larvae completed development and exited incisions as viable third instar larvae. By the end of the experiment (day 8 p.i.) for paired groups T1 and T2, and T3 and T4, healing was not completed in any of the incisions of calves of the control group infested at 14 days p.t. and it was completed in only 75% of the incisions of calves of the control group infested at 21 days p.t. (Fig. 2). Doramectin given 28 days before infestation reduced active lesions by 56%. Incision healing began in two of four animals at 24 h p.i. but two other animals retained infestations with healing patterns that were not different from their corresponding controls. None of the doramectin treated animals exhibited any clinical signs of adverse reaction to medication.

4. Discussion

Results of the first study demonstrated that doramectin gave 100% protection against C. hominivorax infestations induced 2 h after treatment, while ivermectin protected only

50% of the animals exposed to the same challenge. The second study showed that doramectin provided complete protection for 21 days and partial protection (56%) at 28 days p.t. Results of the study designed to compare the efficacy of doramectin and ivermectin against induced challenge of C. hominivorax substantiate initial reports of incomplete efficacy of ivermectin against screwworm (Tregoning, 1993; Benz et al., 1989). This study was conducted under exacting conditions to detect efficacy of treatment. However, even under these conditions 29% of the incisions became infested in ivermectin-treated animals and larvae completed development to adult flies. Recent work by Baez Kohn et al. (1995), indicates that the partial protection of ivermectin is of short duration. When calves, previously treated with doramectin or ivermectin at recommended use levels, were castrated 7 and 15 days later and exposed to natural challenge, the incidence of screwworm myiasis was 60% in control and ivermectin groups and 0% in doramectintreated animals. In the study designed to investigate the end-point of the persistent efficacy of doramectin, 100% protection was obtained for 21 days p.t. This result extends the initial

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findings of Moya-Borja et al. (1993a), who observed protection for 14 days p.t. This sustained efficacy is also consistent with the pharmacokinetic profile of doramectin (Nowakowski et al., 1995). The ability of doramectin to protect susceptible animals against screwworm myiasis in conditions of field challenge has been tested in Latin America (Muniz et al., 1995a,b). The larvicidal activity of doramectin and its persistent efficacy which protects animals for more than 21 days, makes doramectin an ideal tool for control and treatment of screwworm infestations. Because an entire generation of the screwworm-fly is completed in less than 42 days, two injections of doramectin at 21 days interval could prevent larval development in susceptible cattle and reduce screwworm-fly populations in the absence of alternative hosts. Doramectin could also be used when outbreaks occur in previously screwworm-free areas. Susceptible animals can be protected for close to 2 months until enough sterile males are released in the environment to prevent screwworm infestation. The use of a two-dose program of doramectin as described, will also control the most important tropical arthropods and nematode parasites due to its broad spectrum of efficacy (Gonzales et al., 1993; Moya-Borja et al., 1993b; Eddi et al., 1993; Lima et al., 1995). A key aspect of the chemoprophylactic control of C. h o m i n i v o r a x is the possibility to alter traditional husbandry practices, that at present, are limited to times when the parasite is less prevalent in regions where screwworm is endemic (Broce, 1985; Drummond et al., 1988). In those areas, husbandry events such as calving, castration, dehorning, etc., occur only in the winter or in the tropics, the dry season, to avoid the presence of screwworm flies, at a time of the year when nutritional value of the pastures is low. The inflexibility of this management system has two disadvantages. First, it restricts calving to a short time of the year. Second, the conditioning of animals before stressful practices (i.e., castration) and the speed of their recovery, are impaired due to poor nutrition. However, if animals could be adequately protected against C. h o m i n i v o rax, the possibility of calving throughout the year may result in the production of one extra crop of calves during the reproductive life-cycle of a cow. Additionally, husbandry operations could be scheduled at the time of the year most favorable to help prompt return to productivity.

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