Veterinary Parasitology, 37 (1990) 101-I 11 Elsevier Science Publishers B.V., Amsterdam
101
High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep E. Wiedosari 1 a n d D.B. C o p e m a n 2 ~Research Institute for Veterinary Science, P.O. Box 52, Bogor (Indonesia) 2Graduate School of Tropical Veterinary Science, James Cook University, Townsville, Qld., 4811 (Australia) (Accepted for publication 25 April 1990)
ABSTRACT Wiedosari, E. and Copeman, D.B., 1990. High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep. Vet. ParasitoL, 37:101-111. Innate resistance of Javanese thin-tailed sheep to Fasciola gigantica was investigated in animals infected with single doses of 150 or 500 metacercariae and killed 4, 8, 12 or 16 weeks after infection. Infected and non-infected sheep had similar values for packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glutamate dehydrogenase, serum gamma glutamyl transferase and serum aspartate transferase throughout the trial, except for one animal infected with 500 metacercariae from which the highest recovery of flukes ( 55 ) was made. This animal developed pathologically altered values from 12 weeks post infection, coincident with the period of greatest hepatic haemorrhage and destruction of hepatic tissue by migrating flukes and their entry into bile ducts. However, values were altered much less than those reported in other sheep given as few as 200 metacercaria of F. gigantica. Both susceptibility to infection with F. gigantica, as indicated by percentage take of metacercariae and the severity of pathological changes were low in this study in comparison with reports involving other breeds of sheep infected with this parasite. These findings support the conclusion that Javanese thin-tailed sheep have a high innate resistance to F. gigantica.
INTRODUCTION
Fasciolosis is a significant cosmopolitan disease of herbivorous domestic animals, resulting from infection with the trematodes Fasciola hepatica or Fasciola gigantica. In contrast to numerous studies with F. hepatica, little work has been reported on fasciolosis in sheep caused by F. gigantica. There is, nevertheless, some evidence that ovine fasciolosis induced by either species is accompanied by changes in haematological values, especially varying degrees of anaemia, and elevated levels of glutamate dehydrogenase (GD), gamma glutamyl transferase (GGT) and aspartate transferase (AST) in the serum and plasma (Anderson et al., 1977; Sykes et al., 1980; E1 Samani et al., 1985 ). Furthermore, the degree of such changes depends on the intensity of infection and the susceptibility of the host (Sewell, 1966; Reid et al., 1970). 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
102
E. WIEDOSARI AND D.B. COPEMAN
There is also evidence from such measurements as the percentage take after artificial infection, the severity of anaemia and the values of serum/plasma enzymes that some breeds of sheep are more resistant than others to fasciolosis. For example Boyce et al. (1987) found St. Croix and Florida native sheep more resistant to E. hepatica than Merinos (Strong, 1985 ) and Corriedales (Hawkins, 1984). Also, Ogunrinade (1984) found a higher level of resistance to F. gigantica in African dwarf sheep than H a m m o n d (1973) reported in a Merino X Corriedale flock. This study was undertaken as there is no comparable information on the susceptibility of Javanese thin-tailed sheep to fasciolosis and this breed is commonly kept in Indonesia where F. gigantica is a common parasite of domestic ruminants (Soetedjo and Nari, 1980). MATERIALSAND METHODS Nineteen Javanese thin-tailed sheep of both sexes, aged 6-18 months, derived from an area known to be fluke-free were used. Prior to the commencement of the experiment faeces from each animal were examined for trematode eggs by the sedimentation technique with negative results and all sheep were treated with ivermectin to eliminate gastrointestinal nematodes. Throughout the period of observation the sheep were maintained on slats in an animal house and fed concentrate ad libitum plus elephant grass, harvested from an area remote from aquatic snails which was kept stock free. Animals were divided at random into eight groups of two and one of three. Four pairs were infected with 150 and four pairs with 500 metacercariae of F. gigantica. One pair from each group was killed 4, 8, 12 and 16 weeks after infection. The group of three animals was used as control. Blood was drawn from the jugular vein of each animal and transferred to plain tubes and tubes containing sodium EDTA as an anticoagulant, once prior to infection and then every two weeks for 16 weeks. Serum from clotted blood in the plain tubes was separated by centrifugation and stored at - 20 ° C until enzyme measurements were performed. Samples of blood in EDTA tubes were examined haematologically on the day of collection. Metacercariae were produced by infecting Lymnaea auricularia rubigenosa with miracidia hatched from eggs of F. gigantica obtained from infected livers at theBogor abattoir. Prior to infection the metacercariae were examined microscopically for viability and only clear transparent cysts with sharply defined structures were used. Metacercariae were adminstered to sheep as a single oral inoculation enclosed in moist filter paper, using a balling gun. Packed cell volume (PCV) was determined employing a microhaematocrit technique. The Coulter Diluter Dispenser Model DD- 10 (Coulter Electronics Ltd., Northwell Drive, Luton, U.K. ) was used for preparation of samples for
RESISTANCE OF JAVANESE THIN-TAILED SHEEP TO F. GIGANTICA
103
electronic counting of total red cells and total white cells in a Coulter Counter Model ZF and the spectrophotometric determination of haemoglobin using a Coulter haemoglobinometer. The level of GD, G G T and AST in serum was measured using Boehringer M a n n h e i m Kits (Boehringer M a n n h e i m G m b H Diagnostica). Spectrophotometric readings were made at 25°C and 340 nm, 37°C and 405 nm, and 37°C and 340 n m for GD, G G T and AST, respectively. Precinorm and Precipath (Boehringer M a n n h e i m calibrated standards) were used in the quality assurance checks made on all the assays performed. Sheep were killed and flukes were collected from the main bile ducts. The liver was then cut into slices about 5 m m thick and suspended in normal saline while being pressed and squeezed manually to mascerate and liquify the parenchyma. This was washed through a sieve which retained the flukes which were then collected and counted.
Statistical analysis Owing to the removal of animals from the study at various times, the data comprise an unbalanced repeated measures design. The effects included in the model were fixed effects for time, treatments and the interaction between time and treatments and r a n d o m effects of animals within treatments. It follows from the lack of balance that the sums of squares cannot be uniquely attributed to the factors of the model, as would be the case in a balanced design. In such a situation it is most appropriate to consider Type Ill sums of squares; that is the sums of squares are attributed to each factor given all other terms are in the model. All variables were adjusted by substracting their baseline value prior to the application of the treatments. Where significant effects were found the corresponding tables of means are least squares means and not simple marginal means. Least squares means, for unbalanced data, are the equivalent of marginal means for balanced data; they are in effect the means that would have been obtained had the design been balanced. RESULTS
Clinical findings No clinical signs of parasitism were observed in infected sheep, except in the most heavily infected animal from which 55 flukes were recovered 16 weeks after infection. This sheep showed depression, dullness, weakness, loss of appetite and pallor of the mucous membranes 12 weeks after infection. Prior to slaughter at 16 weeks, dyspnoea and abdominal distension were also observed. No deviation from the normal state of health was detected in the control sheep.
104
E, WIEDOSARI AND D.B. COPEMAN
Haematological changes The least squares mean values of PCV, mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration ( M C H C ) above or below pretreatment values in the infected sheep given 150 metacercariae or 500 metacercariae and controls are presented in Figs. 1, 2 and 3, respectively. Values were similar in infected and control sheep throughout the trials, except in one PCV (%) 4
2 O~ -2
-4 -6 "8
f
=
0
2
4
6
8
10
12
14~
16
WEEKS AFTER INFECTION 150 METACERCARIAE
~
5 0 0 METACERCARIAE
~
CONTROL
Fig. 1. Least squares mean values (%) of packed cell volume (PCV) above or below pretreatment values in Javanese thin-tailed sheep after infection with metacercariae ofFasciola gigantica.
MCV 20 r
(FL)
15 ~
'°I I
I
5
-10
0
2
4 WEEKS
150 METACERCARIAE
6
8 AFTER ~
10
12
14
16
INFECTION
5 0 0 MATACERCARIAE
~
CONTROL
Fig. 2. Least squares mean values (FL) of mean corpuscular volume (MCV) above or below pretreatment values in Javanese thin-tailed sheep after infection with metacercariae of Fasciola
gigantica.
RESISTANCE OF JAVANESE THIN-TAILED SHEEP TO F.
4r
105
GIGANTICA
MCHC (G/DL)
2Om -2-4 -6 -8 -10 0
t
i
i
i
t
i
i
2
4
6
8
10
12
14
16
WEEKS AFTER INFECTION 150 METACERCARIAE
~
500 METACERCARIAE
'-'P-- CONTROL
Fig. 3. Least squares mean values (g d1-1 ) of mean corpuscular haemoglobinconcentration (MCHC) aboveor belowpretreatmentvaluesin Javanesethin-tailedsheepafter infectionwith metacercariaeof Fasciolagigantica. individual given 500 metacercariae in which the PCV values dropped sharply 12 and 14 weeks after infection and from which the highest recovery of flukes (55) was made. The values for MCV and MCHC show that the anaemia was normocytic and normochromic. However, there was a terminal macrocytosis (between 12 and 14 weeks) in one sheep given 500 metacercariae. Biochemical values
The least squares mean values above or below pretreatment values of serum GD, and AST after infection of sheep with 150 metacercariae or 500 metacercariae ofE. gigantica and controls are shown in Figs. 4, 5 and 6, respectively. Analysis of the data reveals that as early as 2 weeks after infection, serum GD values were significantly elevated and reached maximum levels 12-14 weeks after infection in sheep given 500 metacercariae (P
101
High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep E. Wiedosari 1 a n d D.B. C o p e m a n 2 ~Research Institute for Veterinary Science, P.O. Box 52, Bogor (Indonesia) 2Graduate School of Tropical Veterinary Science, James Cook University, Townsville, Qld., 4811 (Australia) (Accepted for publication 25 April 1990)
ABSTRACT Wiedosari, E. and Copeman, D.B., 1990. High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep. Vet. ParasitoL, 37:101-111. Innate resistance of Javanese thin-tailed sheep to Fasciola gigantica was investigated in animals infected with single doses of 150 or 500 metacercariae and killed 4, 8, 12 or 16 weeks after infection. Infected and non-infected sheep had similar values for packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glutamate dehydrogenase, serum gamma glutamyl transferase and serum aspartate transferase throughout the trial, except for one animal infected with 500 metacercariae from which the highest recovery of flukes ( 55 ) was made. This animal developed pathologically altered values from 12 weeks post infection, coincident with the period of greatest hepatic haemorrhage and destruction of hepatic tissue by migrating flukes and their entry into bile ducts. However, values were altered much less than those reported in other sheep given as few as 200 metacercaria of F. gigantica. Both susceptibility to infection with F. gigantica, as indicated by percentage take of metacercariae and the severity of pathological changes were low in this study in comparison with reports involving other breeds of sheep infected with this parasite. These findings support the conclusion that Javanese thin-tailed sheep have a high innate resistance to F. gigantica.
INTRODUCTION
Fasciolosis is a significant cosmopolitan disease of herbivorous domestic animals, resulting from infection with the trematodes Fasciola hepatica or Fasciola gigantica. In contrast to numerous studies with F. hepatica, little work has been reported on fasciolosis in sheep caused by F. gigantica. There is, nevertheless, some evidence that ovine fasciolosis induced by either species is accompanied by changes in haematological values, especially varying degrees of anaemia, and elevated levels of glutamate dehydrogenase (GD), gamma glutamyl transferase (GGT) and aspartate transferase (AST) in the serum and plasma (Anderson et al., 1977; Sykes et al., 1980; E1 Samani et al., 1985 ). Furthermore, the degree of such changes depends on the intensity of infection and the susceptibility of the host (Sewell, 1966; Reid et al., 1970). 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
102
E. WIEDOSARI AND D.B. COPEMAN
There is also evidence from such measurements as the percentage take after artificial infection, the severity of anaemia and the values of serum/plasma enzymes that some breeds of sheep are more resistant than others to fasciolosis. For example Boyce et al. (1987) found St. Croix and Florida native sheep more resistant to E. hepatica than Merinos (Strong, 1985 ) and Corriedales (Hawkins, 1984). Also, Ogunrinade (1984) found a higher level of resistance to F. gigantica in African dwarf sheep than H a m m o n d (1973) reported in a Merino X Corriedale flock. This study was undertaken as there is no comparable information on the susceptibility of Javanese thin-tailed sheep to fasciolosis and this breed is commonly kept in Indonesia where F. gigantica is a common parasite of domestic ruminants (Soetedjo and Nari, 1980). MATERIALSAND METHODS Nineteen Javanese thin-tailed sheep of both sexes, aged 6-18 months, derived from an area known to be fluke-free were used. Prior to the commencement of the experiment faeces from each animal were examined for trematode eggs by the sedimentation technique with negative results and all sheep were treated with ivermectin to eliminate gastrointestinal nematodes. Throughout the period of observation the sheep were maintained on slats in an animal house and fed concentrate ad libitum plus elephant grass, harvested from an area remote from aquatic snails which was kept stock free. Animals were divided at random into eight groups of two and one of three. Four pairs were infected with 150 and four pairs with 500 metacercariae of F. gigantica. One pair from each group was killed 4, 8, 12 and 16 weeks after infection. The group of three animals was used as control. Blood was drawn from the jugular vein of each animal and transferred to plain tubes and tubes containing sodium EDTA as an anticoagulant, once prior to infection and then every two weeks for 16 weeks. Serum from clotted blood in the plain tubes was separated by centrifugation and stored at - 20 ° C until enzyme measurements were performed. Samples of blood in EDTA tubes were examined haematologically on the day of collection. Metacercariae were produced by infecting Lymnaea auricularia rubigenosa with miracidia hatched from eggs of F. gigantica obtained from infected livers at theBogor abattoir. Prior to infection the metacercariae were examined microscopically for viability and only clear transparent cysts with sharply defined structures were used. Metacercariae were adminstered to sheep as a single oral inoculation enclosed in moist filter paper, using a balling gun. Packed cell volume (PCV) was determined employing a microhaematocrit technique. The Coulter Diluter Dispenser Model DD- 10 (Coulter Electronics Ltd., Northwell Drive, Luton, U.K. ) was used for preparation of samples for
RESISTANCE OF JAVANESE THIN-TAILED SHEEP TO F. GIGANTICA
103
electronic counting of total red cells and total white cells in a Coulter Counter Model ZF and the spectrophotometric determination of haemoglobin using a Coulter haemoglobinometer. The level of GD, G G T and AST in serum was measured using Boehringer M a n n h e i m Kits (Boehringer M a n n h e i m G m b H Diagnostica). Spectrophotometric readings were made at 25°C and 340 nm, 37°C and 405 nm, and 37°C and 340 n m for GD, G G T and AST, respectively. Precinorm and Precipath (Boehringer M a n n h e i m calibrated standards) were used in the quality assurance checks made on all the assays performed. Sheep were killed and flukes were collected from the main bile ducts. The liver was then cut into slices about 5 m m thick and suspended in normal saline while being pressed and squeezed manually to mascerate and liquify the parenchyma. This was washed through a sieve which retained the flukes which were then collected and counted.
Statistical analysis Owing to the removal of animals from the study at various times, the data comprise an unbalanced repeated measures design. The effects included in the model were fixed effects for time, treatments and the interaction between time and treatments and r a n d o m effects of animals within treatments. It follows from the lack of balance that the sums of squares cannot be uniquely attributed to the factors of the model, as would be the case in a balanced design. In such a situation it is most appropriate to consider Type Ill sums of squares; that is the sums of squares are attributed to each factor given all other terms are in the model. All variables were adjusted by substracting their baseline value prior to the application of the treatments. Where significant effects were found the corresponding tables of means are least squares means and not simple marginal means. Least squares means, for unbalanced data, are the equivalent of marginal means for balanced data; they are in effect the means that would have been obtained had the design been balanced. RESULTS
Clinical findings No clinical signs of parasitism were observed in infected sheep, except in the most heavily infected animal from which 55 flukes were recovered 16 weeks after infection. This sheep showed depression, dullness, weakness, loss of appetite and pallor of the mucous membranes 12 weeks after infection. Prior to slaughter at 16 weeks, dyspnoea and abdominal distension were also observed. No deviation from the normal state of health was detected in the control sheep.
104
E, WIEDOSARI AND D.B. COPEMAN
Haematological changes The least squares mean values of PCV, mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration ( M C H C ) above or below pretreatment values in the infected sheep given 150 metacercariae or 500 metacercariae and controls are presented in Figs. 1, 2 and 3, respectively. Values were similar in infected and control sheep throughout the trials, except in one PCV (%) 4
2 O~ -2
-4 -6 "8
f
=
0
2
4
6
8
10
12
14~
16
WEEKS AFTER INFECTION 150 METACERCARIAE
~
5 0 0 METACERCARIAE
~
CONTROL
Fig. 1. Least squares mean values (%) of packed cell volume (PCV) above or below pretreatment values in Javanese thin-tailed sheep after infection with metacercariae ofFasciola gigantica.
MCV 20 r
(FL)
15 ~
'°I I
I
5
-10
0
2
4 WEEKS
150 METACERCARIAE
6
8 AFTER ~
10
12
14
16
INFECTION
5 0 0 MATACERCARIAE
~
CONTROL
Fig. 2. Least squares mean values (FL) of mean corpuscular volume (MCV) above or below pretreatment values in Javanese thin-tailed sheep after infection with metacercariae of Fasciola
gigantica.
RESISTANCE OF JAVANESE THIN-TAILED SHEEP TO F.
4r
105
GIGANTICA
MCHC (G/DL)
2Om -2-4 -6 -8 -10 0
t
i
i
i
t
i
i
2
4
6
8
10
12
14
16
WEEKS AFTER INFECTION 150 METACERCARIAE
~
500 METACERCARIAE
'-'P-- CONTROL
Fig. 3. Least squares mean values (g d1-1 ) of mean corpuscular haemoglobinconcentration (MCHC) aboveor belowpretreatmentvaluesin Javanesethin-tailedsheepafter infectionwith metacercariaeof Fasciolagigantica. individual given 500 metacercariae in which the PCV values dropped sharply 12 and 14 weeks after infection and from which the highest recovery of flukes (55) was made. The values for MCV and MCHC show that the anaemia was normocytic and normochromic. However, there was a terminal macrocytosis (between 12 and 14 weeks) in one sheep given 500 metacercariae. Biochemical values
The least squares mean values above or below pretreatment values of serum GD, and AST after infection of sheep with 150 metacercariae or 500 metacercariae ofE. gigantica and controls are shown in Figs. 4, 5 and 6, respectively. Analysis of the data reveals that as early as 2 weeks after infection, serum GD values were significantly elevated and reached maximum levels 12-14 weeks after infection in sheep given 500 metacercariae (P
