Veterinary Parasitology, 43 ( 1 9 9 2 ) 2 1 1 - 2 2 2 Elsevier Science Publishers B.V., A m s t e r d a m

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Experiments on anthelmintic control of Fasciola hepatica in Brazil F.A.M. Echevarria a, M.B.C. Correa a, R.D. Wehrle b and I.F. Correa c aEMBRAPA, CP 242, 96400, Bage, Brazil bCiba-Geigy, Basel, CH 4002, Switzerland cCiba-Geigy, Av. Sto. Amaro, 04701, Sao Paulo, Brazil (Accepted 16 December 1991 )

ABSTRACT Echevarria, F.A.M., Correa, M.B.C., Wehrle, R.D. and Correa, I.F., 1992. Experiments on anthelmintic control of Fasciola hepatica in Brazil. Vet. Parasitol., 43:211-222. Two separate field trials involving naturally infected cattle were carried out on two farms known to have a history of Fasciola hepatica infection. On the first farm, 15 animals per group were allocated as follows: G1, triclabendazole (TCBZ) four times a year; G2, TCBZ twice a year (May and September); G3, untreated control. All groups grazed together and after 3.5 years the animals were slaughtered and their livers examined by federal meat inspectors who condemned 100% of livers in G3 and 8.3% in G2 owing to the presence of lesions of fasciolosis. In G 1 no livers were condemned. Significant differences in weight gains were not detected and fluke counts remained at low levels in the treated groups. Also, in the control group, egg counts started to decrease when animals were 2 years old. On the second farm, groups of 20 animals were treated as follows: G 1, TCBZ three times a year (May, September and December); G2, TCBZ twice a year (May and September); G3, nitroxynil twice a year (May and September ); G4, rafoxanide twice a year (May and September); G5, untreated controls. All animals were weighed and faecal samples examined at approximately 28-day intervals. During the period of the study, larger weight gains were detected in the TCBZ treated groups than in the others. TCBZ treatment kept F. hepatica egg counts at a lower level for longer periods than the other drugs and significant differences in weight gains were only obtained between the group receiving TCBZ three times a year and the control group.

INTRODUCTION

Fasciolosis is one of the most important parasitic diseases in animals of economic importance worldwide. This is mainly due to losses associated with condemnation of livers, mortality, reduction in production of meat, wool and milk, by secondary bacterial infections, by interference with fertility rates and by the costs of anthelmintic treatments. In southern Brazil, in the state of Rio Grande do Sul, about 16% of the slaughtered animals have their livers conCorrespondence to: F.A.M. Echevarria, E M B R A P A , CP 242, 96400, Bage, Brazil.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0 3 0 4 - 4 0 1 7 / 9 2 / $ 0 5 . 0 0

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demned by the federal meat inspectors and this represents losses of US$ 400 000 year-~ to the meat industry (Ministry of Agriculture, unpublished data, 1987). This paper reports the results obtained in two experiments with beef cattle where different fluke treatments were applied from weaning up to slaughter of animals acquiring natural infections under field conditions. MATERIALS AND METHODS

Two farms, known to have cattle infected with Fasciola hepatica, were chosen for the trials: Experiment 1 at county Bage and Experiment 2 at county D o m Pedrito. In Experiment l, 45 Hereford and Hereford/Limousin 10-month-old calves, weaned and naturally infected with F. hepatica were ordered according to body weight and allocated at random to the following treatments. GI: triclabendazole ( 12 mg kg-~ ) in August 1984 and from then on every 3 months (TCBZ 4Xyear-~); G2: triclabendazole (12 mg kg -1 ) in August 1984 and from May 1985, in May and September each year (TCBZ 2 Xyear-1 ); G3: control (untreated). In Experiment 2, 100 Hereford and Hereford/Holstein calves 6-8 months of age, weaned and naturally infected with F. hepatica were allocated, as in Experiment 1, to five treatments as follows. G 1: triclabendazole ( 12 mg kg- ~) in May, September and December starting in 1985 until slaughter (TCBZ 3 × year- ~); G2: triclabendazole ( 12 mg kg -~ ) in May and September starting in 1985 until slaughter (TCBZ 2 X year- ~); G3: nitroxynil ( 10 mg kg-~ ) in May and September starting in 1985 until slaughter (NTX 2 × year- ~); G4: rafoxanide (3 mg kg -~ ) in May and September starting in 1985 until slaughter (RFX 2 >(year- ~); G5 control (untreated). Triclabendazole was administered per os whereas nitroxynil ( N T X ) and rafoxanide (RFX) were given as subcutaneous injections. In the group treated with RFX, a subcutaneous formulation (3 mg kg ~) was used until the middle of the experiment; as it was withdrawn from the market the oral formulation (7.5 mg kg-~ ) was used from then on. All animals were treated against gastrointestinal nematodes following recommendations for the locality which involved four treatments per year (Pinheiro, 1983). The drugs used were fenbendazole and levamisole which have no flukicidal activity. Although the above recommendations apply to animals of up to 2 years of age, in the present studies the experimental animals contin-

ANTHELMINTIC CONTROL OF FASCIOLA HEPATICA IN BRAZIL

2]3

ued to be treated at the same levels until the end of the experiments in order to reduce any parasitic effect other than the effect of fluke parasitism. At approximately 28-day intervals all animals were weighed and their faeces sampled for fluke egg count determination (Girao, 1982). The animals at both experimental sites were kept under conditions of extensive grazing on natural unimproved pastures and control and treated groups were grazed together as a whole herd. Once the animals reached the appropriate weight (460-480 kg) they were sent to an abattoir where they were slaughtered and weights of the dressed carcass recorded both at room temperature (gross carcass weight ) and after 24 h exposure to - 10 °C (net carcass weight ). After being examined by the federal meat inspectors, all livers were taken to the Laboratory of Parasitology at Embrapa's Research Station in Bage where they were screened for individual fluke burdens. Analysis of variance was used on all data and means were compared using Fisher's least significance test. Fluke numbers in Experiment 2 were transformed to log~0 ( c o u n t + 1 ) before being submitted to one-way analysis of variance and the Newman-Keuls multiple range test. RESULTS

Fasciola hepatica faecal egg counts Fluke egg counts are shown in Figs. 1-3. In Experiment 1, counts in the control group rose sharply and remained high until animals were 2 years old, when they dropped to low levels (Fig. 1 ). In Experiment 2, the highest counts were only detected in the control group when animals reached the age of 2 years but all treated groups had their highest egg counts soon after the start of the experiment.

Weight gains Accumulated weight gains in the two experiments under different treatments are shown in Figs. 4-6. In Experiment 1, although a difference of 13.4 kg was seen between the TCBZ 4 × year- l group and the control group, this difference was not statistically significant (Table 1 ). On the other hand, in Experiment 2 there was a significant difference ( P < 0.05 ) of 31.3 kg between the TCBZ 3 X year-~ group and the control group but no significant differences were detected between the other treatments (Table 2). Mean daily weight gains (g day- l ) in Experiment 1 ranged between 293 _+33.9 g for TCBZ 2 × y e a r -1 and 280___39.4 g for the control and did not differ significantly ( P > 0.05 ). In Experiment 2 a significant difference ( P < 0.05 ) was obtained only between the TCBZ 3 >(year-l group (257 + 29.0 g) and the control group ( 2 3 5 ± 3 0 . 0 g ) (Table 2).

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ANTHELM1NTIC C O N T R O L OF FASCIOLA tlEPATIC4 IN BRAZIL

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ANTHELMINTIC CONTROL OF FASCIOLAHEPATICAIN BRAZIL

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TABLE1 Total weight gains, daily weight gains, carcass weights and percentages of condemned livers in Experiment 1

Total weight gain (kg) Daily weight gain (g) Gross carcass weight t (kg) Net carcass weight2 (kg) Loss by cold exposure (%) Condemnedlivers ( n/x) (%)

TCBZ 4 × year- ~

TCBZ 2 × year-

Control

385.0 291.0 241.8 236.7 2.1 0

382.7 293.0 239.7 233.5 2.6 8.3

371.6 280.0 243.3 238.5 1.9 100.0

(___45.4) a (_+34.3) a (+_26.8) a (_+25.5) a (_+2.3) a (0/12)

(+_48.4)" (+-33.9)" (+-40.6)" (_+40.1) a (_+1.5) a (1/12)

(_+52.1) a (+-39.4)" (_+41.3) a (_+40.3) a (+_0.9)" (8/8)

~Weight at room temperature. 2Weight after 24 h exposure to - 10 ° C. Values within a row followed by the same superscript are not significantly different ( P > 0.05).

Carcass No significant differences ( P > 0.05 ) were obtained in gross carcass weight, net carcass weight or in losses due to exposure to cold between the different treatments (Tables 1 and 2). Water losses in carcasses exposed to - 10°C in Experiment 1 varied between 1.9 _+0.9% in the control group and 2.6 _+ 1.5% in the TCBZ 2 X y e a r - ' group, whereas in Experiment 2 the losses were around 2.3% with the exception of the group treated with RFX which lost 3.6 _+5.0%.

Livers When examined by the federal meat inspectors all livers in Experiment 1 in the TCBZ 4 × y e a r - 1 group were found fit for h u m a n consumption and only one ( 1/ 12) was c o n d e m n e d in the TCBZ 2 X y e a r - ' group whereas all livers in the control group were c o n d e m n e d owing to the presence of F. hepatica or associated lesions (Table 1 ). No flukes were found in the groups treated with TCBZ (Table 3) while in the control 3.6 flukes per liver were detected but two livers had only chronic lesions and calcified flukes. All livers in Experiment 2 were c o n d e m n e d for h u m a n consumption owing to the presence of liver fluke a n d / o r associated lesions (Table 2). The smallest fluke burden (0.6) was found in the TCBZ 3 × y e a r - ~ group where only three livers were parasitised, the other being c o n d e m n e d for the presence of lesions. With the exception of the groups treated with R F X and NTX, all treated groups had different fluke burdens ( P < 0.05 ) from that of the control group. There was no significant difference in fluke burdens between TCBZ 2 X y e a r - l, N T X 2 X y e a r - ' and R F X 2 × y e a r - ~groups (Table 3 ). It is also important to mention that during the development of both trials, anaplasmosis and babesiosis, but not fasciolosis, caused the death of some

363.1 257.0 225.7 220.5 2.3 100

(_+40.1) a ( + 29.0)" (_+26.6) ~ (+26.2) a (+0.5) a (14/14)

~ 354.3 250.0 216.3 211.6 2.2 100 -+36.5) ~'b _+25.1) ~'b +25.9) a -+25.2) a _+0.5)a 19/19)

TCBZ 2 × y e a r -~ 353.9 (_+33.8) ab 2 5 0 . 0 ( + 2 3 . 9 ) ab 217.6 ( + 2 3 . 7 ) " 212.7 (_+23.9) a 2.3 ( + 1.2)" 100 ( 1 7 / 1 7 )

NTX 2 × y e a r -~

~Weight at room temperature. eWeight after 24 h exposure to - 10:C. Values within a row followed by' different superscripts are significantly different ( P < 0.05 ).

Total weight gain (kg) Daily weight gain (g) Gross carcass weight t (kg) Net carcass weight z (kg) Loss by cold exposure (%) Condemned livers (n/x) (%)

TCBZ 3×year

Total weight gains, daily weight gains, carcass weights and percentages o f c o n d e m n e d livers in Experiment 2

TABLE 2

352.2 (_+33.1) "~' 2 5 0 . 0 ( + 2 3 . 5 ) ab 219.7 (_+ 15.7) a 212.2 ( + 2 1 . 7 ) a 3.6 (_+ 5.0) a 100 ( 1 8 / 1 8 )

RFX 2 × y e a r -* 331.8 235.0 215.6 210.1 2.6 t00

(_+42.4) b ( + 30.0) b (_+21.9)" (_+21.5) a (+0.6) a (16/16)

Control

r"-

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A N T H E L M I N T I C C O N T R O L O F FASCIOLA HEPATICA IN BRAZIL

219

TABLE 3 Number ofF.

hepatica found at slaughter o f animals in Experiments 1 and 2

Experiment

Treatment

n

No. o f flukes per liver

Mean

1

TCBZ 4 × y e a r ~ TCBZ 2 × y e a r - t Control TCBZ 3 × y e a r t TCBZ 2 × y e a r -~ N T X 2 × y e a r -~ RFX 2 ×year -I Control

12 12 8 14 19 17 18 16

0,0,0,0,0,0,0,0,0,0,0,0 0,0,0,0,0,0,0,0,0,0,0,0 1,7,0,9,5,2,0,5 2,0,0,0,0,3,3,0,0,0,0,0,0,0 8,2,3,16,0,0,6,0,0,0,5,3,8,1,0,0,2,7,2 8,12,0,0,11,8,5,2,7,1,3,3,7,1,2,0,0 0,45,2,7,12,31,4,0,0,7,2,7,5,1,4,3,0,7 13,4,16,20,8,0,30,10,4,1,7,8,2,25,3,8

0 0 3.6 0.6" 3.3 b 4.1 bc 7 . 6 bc

9.9 c

a,b,CMeans with different superscripts are significantly different ( P < 0.05 ).

experimental animals, and this is the reason for the different group sizes shown in Tables 1-3. DISCUSSION

In order to detect possible effects of fasciolosis in cattle, production investigations should be carried out over long periods of time (Boray, 1963); the experiments reported in this paper were conducted beginning with young animals (less than 1 year old) until they reached the appropriate slaughter weight, which under the conditions of extensive grazing management was only possible when they reached 4.5 years of age. Both experiments were conducted on farms known to have a history of having animals heavily infected with F. hepatica and although statistical comparisons between the two experiments were not carried out, as they had different experimental designs, their results showed a different trend in fluke egg counts. In Experiment 1, the control group reached high fluke egg levels in the faeces (Fig. 1 ) soon after the start of the trial. This experiment started in August (end of winter) when the climatic conditions start to become more favourable for the development of the intermediate hosts Lymnaea columella and Lymnaea viatrix. Experiment 2 started in autumn (May) when the lower temperatures are not suitable for the snails; because of this, the rise in fluke egg counts was slower, only reaching its highest level when the animals were 2 years old in the second spring (September). The fall in fluke burden suggested by the sharp fall in fluke egg counts detected in Experiment 1 (Fig. 1 ) when the animals reached 2 years of age may be due to two factors. First, as all groups (treated and controls) grazed together and one-third of the animals were treated every 3 months and the other one-third twice a year with TCBZ, which is highly efficient against immature stages (Boray et al., 1983) these animals may have produced a smaller con-

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F.A.M. ECHEVARRIA ET AL.

tamination of the experimental paddock, reducing in this way the probability of reinfections by the control animals. Nevertheless, this is just a hypothesis as no quantitative examination of snails was carried out in either of the experimental sites. Secondly, the reduced contamination, and in consequence a lower but continuous challenge, might have contributed to the development of immunity to further reinfections. As a consequence of these two factors no significant differences were detected on the productive aspects studied as weight gains and carcass weight. In the second experiment where only two groups were treated with TCBZ, and at longer intervals than in Experiment 1, and where the other flukicides used, NTX and RFX have lower efficacy on immature stages than TCBZ (Boray et al., 1983), there was a more gradual rise in the level of infection (as measured by fluke egg counts) in the control group until animals were 2 years old, when it started to decline gradually until the age of 3.5 years when an accentuated drop occurred (Figs. 2 and 3 ). The fact that the controls in Experiment 2 reached their highest egg counts only when they were 2 years old, together with the detection of higher egg counts in all treated groups since the beginning of the experiment, compared with the controls (Figs. 2 and 3 ), may suggest a lack of development of immunity up to that age. The less frequent treatments with TCBZ plus the use of flukicides of lower efficacy in Experiment 2 probably resulted in a more continuous contamination of the experimental area with eggs which in turn led to more snail infection and higher fluke burden reflected in detection of significant differences in weight gains between the TCBZ 3 X year-~ group and the control group. On the other hand, a constant contamination of the area and the larger interval between treatments did not allow the detection of significant differences between the 2 X year- l groups and the control group. The greater efficacy of TCBZ compared with the other flukicides ( Boray et al., 1983) was also detected in Experiment 2 where the group treated with that drug in May and September showed lower levels of fluke egg counts in comparison with the other drugs applied at the same time (Figs. 2 and 3). This greater efficacy led to a 22.5 kg difference in liveweight over the control, although this was not significant ( P > 0.05 ). Higher water losses in carcasses exposed to - 10°C during 24 h may be an indication of oedema, which can occur in acute fluke infections. In the present study, by the time of slaughter animals were carrying chronic infections ofF. hepatica and no significant differences were detected. Also, the time and intensity of the effects of fasciolosis on liveweight are dependent on two variables - - fluke burden and nutritional level (Kendall et al., 1978) the main effects being observed between 12 and 16 weeks after artificial infection (Hope-Cawdery et al., 1977). On the other hand, most of the experiments carried out so far have used artificial infections and, in some, the results are conflicting. Burden et al. (1978) using calves which were housed, well fed

ANTHELMINTIC CONTROL OF FASCIOLA fIEPATI(14 IN BRAZIL

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and artificially infected, obtained good weight gains even with burdens of 440 flukes but reduced gains and severe losses were detected when calves remained at grass until the end of a u t u m n in European conditions (Reid et al., 1972). In the two experiments reported here, where results were obtained with naturally acquired infection and where the nutrition level was always low, especially during the winter periods, the numbers of flukes found in the control groups were low. Nevertheless, high egg counts were detected at the beginning of the trial which led to lower weight gains in the control group in Experiment 2 since the first month. This difference in weight gain became more pronounced when the animals reached the age of 1.5 years, and by the end of the trial a difference of 8.6% over the control group was achieved, which is within the range of 8.4-25.9% obtained by Fromunda et al. ( 1975 ). The migration of a small number of flukes in the liver in cattle can occur without clinical signs or impaired weight gains (Sinclair, 1967). Nevertheless, the lesions caused by this migration leads to condemnation of livers for h u m a n consumption at the abattoirs as once the hyperplastic cholangitis and hepatic fibrosis have developed the changes are irreversible (Echevarria et al., 1979 ). In Experiment 1 where only TCBZ was used, all livers were healthy with only one liver being condemned in the TCBZ 2 × year- 1 group, but in Experiment 2, although there were significant differences in weight gains, the presence of lesions or parasites led to condemnation of all the livers. The results of these experiments confirm the high efficacy of triclabendazole as a flukicide and suggest that it would be the drug of choice to be used in a control programme aimed at reducing the effects of F. hepatica in cattle. Nevertheless, under conditions of extensive grazing management prevailing in southern Brazil, even this highly efficient drug may not be able to prevent some liver condemnation. ACKNOWLEDGEMENTS

The authors are grateful to Dr. J.G.C. da Silva for the help with the statistical analysis and to Professor J. Armour for reviewing this report. A. Azambuja and E. Galarza provided facilities and animals at their farms and this is greatly appreciated. Both experiments were supported by Ciba-Geigy for which we are grateful.

REFERENCES Boray, J.C., 1963. Standardization of techniques for pathological and anthelmintic studies with Fasciola spp. In: Proceedings of the International Conference of the World Association for the Advancement of Veterinary Parasitology, Vol. 1, Hannover, 1963, pp. 34-35. Boray, J.C., Crowfoot, P.D., Strong, M.B., Allison, J.R., Schellenbaum, M., von Orelli, M. and

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Sarasin, G., 1983. Treatment of immature and mature Fasciola hepatica infections in sheep. Vet. Rec., 113: 315-317. Burden, D.J., Hughes, D.L., Hammet, N.C. and Colis, K.A., 1978. Concurrent daily infection of calves with Fasciola hepatica and Ostenagia ostertagq. Res. Vet. Sci., 25: 302-306. Echevarria, F.A.M., Pinheiro, A.C. and Alves-Branco, F., 1979. Tratamento da fasciolose cronica em bovinos. Pesqui. Agropec. Bras., 14:185-188. Fromunda, V., Minascurta, C. and Popescu, S., 1975. Essai du nitroxynil (Dovenix) dans le traitement de la fasciolose bovine en Roumanie. Cah. M6d. V6t. 44:151-154. Girao, E.S., 1982. Tecnica de quatro tamises para o diagnostico coprologico quantitativo de fasciolose dos ruminantes. M.Sc. Thesis, Veterinary Faculty, Universidade Federal do Rio Grande do Sul. Hope-Cawdery, M.J., Strickland, K.L., Conway, A. and Crowe, P.J., 1977. Production effects of liver-fluke in cattle. 1. The effects of infection on liveweight gain, feed intake and food conversion efficiency in beef cattle. Br. Vet. J., 133:145-159. Kendall, S.B., Sinclair, l.J., Everett, G. and Parfitt, J . W . , 1978. Resistance to Fasciola hepatica in cattle. I. Parasitological and serological observations. J. Comp. Pathol, 88:115-122. Pinheiro, A.C., 1983. Programas de controle de verminose-- Aspectos tecnicos e relacao custobeneficio. In: Jornadas sobre producao de novilho jovem, Bage, pp. 65-73. Reid, J.F.S., Doyle, J.J., Armour, J. and Jennings, F.W., 1972. Fasciola hepatica infection in cattle. Vet. Rec., 90: 486-487. Sinclair, K.B., 1967. Physiological aspects of fascioliasis. In: Int. Liverfluke Colloq., 2: 157163.