Veterinary Parasitology, 35 (1990) 157-161 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
157
Short C o m m u n i c a t i o n
T h e S u s c e p t i b i l i t y of L y m n a e i d Snails to Paramphistomum cervi Infections in M e x i c o L. CASTRO-TREJO, Z. GARCJA-VASQUEZ* and J. CASILDO-NIETO
Centro Nacional de Parasitologia - - SARH, Apartado Postal 206, CIVAC C.P. 62500 Morelos (Mexico) (Accepted for publication 30 March 1989)
ABSTRACT Castro-Trejo, L., Garcla-Vasquez, Z. and Casildo-Nieto, J., 1990. The susceptibility of Lymnaeid snails to Paramphistomum cervi infections in Mexico. Vet. Parasitol., 35: 157-161. The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.
INTRODUCTION
Paramphistomiasis of sheep and cattle is a disease causing serious economic losses to the wool, meat and milk industries. During 1965, at least 64 cattle deaths were attributed to this disease in South Africa (Horak, 1967 ) and Boray (1959) mentioned a cattle mortality rate of 40%. However, little attention has been paid to this disease in Mexico. There are a few reports on the occurrence of Paramphistomum cervi in Mexico in sheep (Quiroz and Ochoa, 1972) and cattle (Campos et al., 1983), although other paramphistome species in cattle such as Cotylophoron cotylophorum and Calicophoron have been identified (de la O et al., 1983). The intermediate hosts of P. cervi are freshwater snails, which rarely leave water. In certain drought conditions, a few snails can survive by aestivation. Infection occurs when animals graze or drink water near the habitat of infected snails. Several genera of snails, such as Bulinus spp., Glyptanisus gilberti, Indoplanorbis exustus, Planorbis planorbis and Lymnaea bulimoides have been *Author to whom correspondence should be addressed.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
158
L. CASTRO-TREJO ET AL.
reported to serve as intermediate hosts of P. cervi in different parts of the world (Soulsby, 1974 ). The snails of the genus Lymnaea are widely distributed throughout the coastal regions of Mexico (Castro-Trejo and Casildo-Nieto, 1984). In an infection study, the susceptibility of five different species of this snail to P. cervi was examined under laboratory conditions and the results are reported here. MATERIALSAND METHODS
Snails Five species of the genus Lymnaea were originally collected from different locations in Mexico and identified by their characteristics: the shell is dextral and ovately oblong, and varies in thickness and size of the body whorl; the spire is more-or-less attenuated and varies considerably in height; the columellar axis is typically twisted; the central tooth of the radula and the lateral teeth are distinctive. They were designated according to their origins as follows: L. bulimoides (Tulancingo, Hgo. ), L. columella (Huachinango, Pue. ), L. cubensis (Tetecala, Mor.), L. humilis (Tuxpan, Ver.), L. palustris (Xochitepec, Mor). They were reared and bred in the laboratory according to the method of Taylor and Mozley (1948) using glass Petri dishes (100 X 20 mm) containing microalgae of the genus Oscilatoria and mud as the maintenance medium. Laboratory-bred, parasite-free specimens were used for exposure with miracidia throughout the experiment. Paramphistomum cervi miracidia Adult parasites of P. cervi were collected from the rumen of cattle in an abattoir in Tuxpan Ver. These were washed and incubated at 37 °C in Petri dishes with tap water for 24 h. The eggs laid by the parasites were collected by sedimentation and incubated at 29 ° C for 13 days. These were later exposed to light to facilitate hatching and the release of miracidia.
Injection of snails Batches of 1100 snails of each of the five species were used for exposure to miracidia. From each batch, 11 groups of 100 snails were formed, one group was left as an uninfected control and the other groups were exposed to 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 miracidia. The snails were exposed to miracidia individually according to the method of Kendall and Parffit (1959) with slight modifications. Briefly, 96-well polystyrene microtiter plates were used for holding the snails. Each well was filled with distilled water, a single snail deposited in it and a defined number of
INFECTION OF LYMNAEIDS WITH P A R A M P H I S T O M U M CERVI
159
miracidia applied. After 3 h of contact with the miracidia at room temperature, the snails were transferred to Petri dishes containing the maintenance medium and maintained at room temperature (24-27 °C ). Thirty days post-infection, each exposed snail was deposited in a 100-ml glass beaker containing a piece of yellow wax paper and water. The beaker was put in an ice-water bath to stimulate the emergence of cercariae; the latter encysted on the yellow wax paper as described by Durie (1955). The metacercariae were counted. The snails which did not shed any cercariae or which were dead were dissected to determine if they harboured the larval stage of the trematode.
Infection of sheep To confirm the identity of the isolated metacercariae, they were stored for 40 days, pooled and used for infecting Pelibuey sheep which were reared, housed and maintained in paramphistome-free conditions. Ten 2-year-old sheep were divided into five groups of two animals. Each group was infected orally with 50, 100, 500, 1000 or 5000 metacercariae. One sheep from each group was slaughtered at 70 days and the other 180 days post-infection. At necropsy, the paramphistomes were recovered from the rumen and counted. A representative number of recovered worms was relaxed, flattened between two slides and fixed in normal saline containing 10% formalin. They were stained by Gomori's stain and mounted for specific identification according to Dinnik (1961) and Kotrla and Prokovic (1973). RESULTS Of the five lymnaeid species exposed to P. cervi miracidia, only three species, L. cubensis, L. humilis and L. palustris, picked up the infection and L. bulimoides and L. columella proved refractory. A larger proportion of the exposed L. palustris was infected and shed cercariae than L. cubensis and L. humilis (Table 1 ). An infection dose of 6 miracidia produced 100% infection in L. palustris, whereas an exposure dose of > 12 miracidia was necessary to produce a 50% infection rate in the other two snail species; the rate of infection in these two species never reached 100% (Table 1 ). The mortality of the exposed L. palustris increased with an increase in the exposure dose with miracidia. It was higher in the case of L. palustris than in L. hurnilis and L. cubensis although, in relative terms, the latter two species showed a similar rate of mortality to the former in relation to their rate of infection. In the three species of Lymnaea successfully infected with P. cervi and surviving for 35 days post-infection and shedding cercariae, the number of cercariae discharged was inversely related to the miracidial exposure dose; the
160
L. CASTRO-TREJO ET AL.
'FABLE 1
Susceptibility, mortality and cercarial emergence from L. cubensis, L. humilis and L. palustris exposed to different numbers of Paramphistomum miracidia 1 Miracidia dose
2 4 6 8 10 12 14 16 18 20
Control
L. cubensis
L. humilis
L. palustris
No.
No.
No.
No.
No.
No.
No.
No.
infected
dead
shed
infected
dead
shed
infected
dead
25 30 41 40 45 57 59 60 60 60 0
1 4 4 5 14 33 58 60 60 60 0
3 4 2 1 0 0 0 -
13 25 33 35 35 39 47 57 53 58 0
8 1 1 4 14 27 34 53 49 56 0
1 1 1 1 1 0 0 0 0 0 0
89 98 100 100 100 100 100 100 100 100 0
0 2 2 24 79 99 92 95 100 100 0
0
No. shed 65 55 30 9 1 0 0 0
0
1Eleven groups of 100 snails each.
snail infected with two miracidia shed a high number of cercariae and the number decreased with an increase in the exposure dose. The sheep in all five groups, infected orally with different doses of metacercariae, showed the presence of P. cervi in their rumens. The sheep slaughtered at 70 days post-infection showed only juvenile parasites, whereas at 180 days post-infection, both juvenile and adult parasites were recovered, though the relative proportion of adults were less than juveniles. In both the instances, the number of worms recovered was dependent on the level of infection dose. DISCUSSION
AND CONCLUSION
The epidemiology of paramphistomiasis has not been studied exhaustively in Mexico. The snails of the genus Lymnaea spp., which serve as the intermediate host of Fasciola hepatica, are widely distributed in different regions of Mexico where cattle and sheep are raised (Castro-Trejo et al., 1983). This study shows that L. cubensis, L. humilis and L. palustris may act as intermediate hosts of P. cervi in Mexico. L. bulimoides and L. columeUa, were earlier reported to be intermediate hosts of P. cervi (Olsen, 1974), but in this study, both these species were refractory to experimental infections. L. palustris seems to maintain the parasite in nature and may help propagate the infection in Mexican ruminants. When snails were infected with fewer miracidia, they shed large numbers of cercariae; this has epizootiological implications. The other two lymnaeid species, L. cubensis and L. humilis could also be infected with P. cervi under laboratory conditions and, from the epizootiolog-
INFECTIONOFLYMNAEIDSWITHPARAMPHISTOMUMCERVI
161
ical point of view, these may appear to be less effective transmitters of the trematode. The maturation of the metacercariae to immature and mature stages of adult P. cervi in sheep confirmed that the cercariae shed by the infected snails, were viable and that the molluscan phase of the life cycle of the parasite was successfully completed. Traditionally, the introduction of Pelibuey sheep to cattle farms is a common practice in the tropical regions of Mexico. This situation offers an opportunity for the introduction and propagation of parasites in cattle, especially where the Lymnaea species are abundant. In conclusion, three species of Lyrenaea snails have been found to act as intermediate hosts of P. cervi under experimental conditions. To discover more about the epizootiology of paramphistomiasis, it is necessary to investigate further the relative distribution of various species of Lymnaea as well as the prevalence of snails infected with P. cervi in Mexico. REFERENCES Boray, J.C., 1959. Studies on intestinal amphistomosis in cattle. Aust. Vet. J., 35:282 287. Campos, R.R., Escutia, S.I. and Herrera, R.D., 1983. Estudio epizootiologico de algunas parasitosis internas de bovinos en el Istmo de Tehuantepec. In: Una ddcada de investigacidn en el Departamento de Parasitologia ( 1972-1982 ). Instituto Nacional de Investigaciones Pecuarias, SARH, Mexico, 64 pp. Castro-Trejo, L., Mata, E., Gonzalez, O. and Garcfa-Vasquez, Z., 1983. Estudio integrado de la Fasciolasis Bovina en el Estado de Durango. Memorias. Reunion de Investigaciones Pecuarias, SARH, M~xico, pp. 254-257. Castro-Trejo, L. and Casildo-Nieto, J., 1984. Vectores de la distomatosis hepatica an M~xico. IV. Congreso Nacional de Parasitologia Minatitlan Ver. M6xico, 58 pp. De la 0., J.R., Acevedo, A.H. and Quintero, M., 1983. Frecuencia y determinaci6n de trematodos de la fhmilia Paramphistomatidae (Fischoeder, 1901 ) de bovinos sacrificados en el frfgorifico y empacadora de Tabasco, S.A., Villahermosa Tabasco. Mere. Reun. An. Parasitol. Vet., Mexico, 16 pp. Dinnik, J.A., 1961. Paramphistomum phillerouxi (Trematoda): Paramphistomatidae and its development in Bulinus forskalli. J. Helminthol., 35: 69-90. Durie, P.H., 1955. A technique for collection of large number of paramphistome (Trematoda) metacercariae. Aust. J. Agric. Res., 6: 200-202. Horak, T.G., 1967. Host-parasite relationship of Paramphistomum microbothrium Fischoeder, 1901 in experimentally infected ruminants with particular reference to sheep. Ondersterpoor. J. Vet. Res., 34: 451-540. Kendall, S.B. and Parffit, J.W., 1959. Studies on the susceptibility of some species of Lymnaea to infection with Fasciola gigantica and F. hepatica. Ann. Trop, Med. Parasitol., 53: 220-227. Kotrla, B. and Prokovic, J., 1973. Paramphistomiasis of cattle in Cuba. Acta Vet., 42:35 44. Olsen, O.W., 1974. Animal Parasites, Their Life Cycles and Ecology. 3rd edn. University Press, pp. 323-342. Quiroz, H. and Ochoa, R., 1972. Presencia de Paramphistomum cervi (Schrank 1790 ), en un ovino de raza Tabasco 6 Pelibuey en M6xico. Tec. Pec. Mex., 21: 59. Soulsby, E.J., 1974. Helminths, Arthropods and Protozoa of Domestic Animals. Williams & Wilkins, Baltimore, MD, 64 pp. Taylor, E.L. and Mozley, A., 1948. A culture method for Lymnaea truncatula. Nature (London), 161: 594.
157
Short C o m m u n i c a t i o n
T h e S u s c e p t i b i l i t y of L y m n a e i d Snails to Paramphistomum cervi Infections in M e x i c o L. CASTRO-TREJO, Z. GARCJA-VASQUEZ* and J. CASILDO-NIETO
Centro Nacional de Parasitologia - - SARH, Apartado Postal 206, CIVAC C.P. 62500 Morelos (Mexico) (Accepted for publication 30 March 1989)
ABSTRACT Castro-Trejo, L., Garcla-Vasquez, Z. and Casildo-Nieto, J., 1990. The susceptibility of Lymnaeid snails to Paramphistomum cervi infections in Mexico. Vet. Parasitol., 35: 157-161. The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.
INTRODUCTION
Paramphistomiasis of sheep and cattle is a disease causing serious economic losses to the wool, meat and milk industries. During 1965, at least 64 cattle deaths were attributed to this disease in South Africa (Horak, 1967 ) and Boray (1959) mentioned a cattle mortality rate of 40%. However, little attention has been paid to this disease in Mexico. There are a few reports on the occurrence of Paramphistomum cervi in Mexico in sheep (Quiroz and Ochoa, 1972) and cattle (Campos et al., 1983), although other paramphistome species in cattle such as Cotylophoron cotylophorum and Calicophoron have been identified (de la O et al., 1983). The intermediate hosts of P. cervi are freshwater snails, which rarely leave water. In certain drought conditions, a few snails can survive by aestivation. Infection occurs when animals graze or drink water near the habitat of infected snails. Several genera of snails, such as Bulinus spp., Glyptanisus gilberti, Indoplanorbis exustus, Planorbis planorbis and Lymnaea bulimoides have been *Author to whom correspondence should be addressed.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
158
L. CASTRO-TREJO ET AL.
reported to serve as intermediate hosts of P. cervi in different parts of the world (Soulsby, 1974 ). The snails of the genus Lymnaea are widely distributed throughout the coastal regions of Mexico (Castro-Trejo and Casildo-Nieto, 1984). In an infection study, the susceptibility of five different species of this snail to P. cervi was examined under laboratory conditions and the results are reported here. MATERIALSAND METHODS
Snails Five species of the genus Lymnaea were originally collected from different locations in Mexico and identified by their characteristics: the shell is dextral and ovately oblong, and varies in thickness and size of the body whorl; the spire is more-or-less attenuated and varies considerably in height; the columellar axis is typically twisted; the central tooth of the radula and the lateral teeth are distinctive. They were designated according to their origins as follows: L. bulimoides (Tulancingo, Hgo. ), L. columella (Huachinango, Pue. ), L. cubensis (Tetecala, Mor.), L. humilis (Tuxpan, Ver.), L. palustris (Xochitepec, Mor). They were reared and bred in the laboratory according to the method of Taylor and Mozley (1948) using glass Petri dishes (100 X 20 mm) containing microalgae of the genus Oscilatoria and mud as the maintenance medium. Laboratory-bred, parasite-free specimens were used for exposure with miracidia throughout the experiment. Paramphistomum cervi miracidia Adult parasites of P. cervi were collected from the rumen of cattle in an abattoir in Tuxpan Ver. These were washed and incubated at 37 °C in Petri dishes with tap water for 24 h. The eggs laid by the parasites were collected by sedimentation and incubated at 29 ° C for 13 days. These were later exposed to light to facilitate hatching and the release of miracidia.
Injection of snails Batches of 1100 snails of each of the five species were used for exposure to miracidia. From each batch, 11 groups of 100 snails were formed, one group was left as an uninfected control and the other groups were exposed to 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 miracidia. The snails were exposed to miracidia individually according to the method of Kendall and Parffit (1959) with slight modifications. Briefly, 96-well polystyrene microtiter plates were used for holding the snails. Each well was filled with distilled water, a single snail deposited in it and a defined number of
INFECTION OF LYMNAEIDS WITH P A R A M P H I S T O M U M CERVI
159
miracidia applied. After 3 h of contact with the miracidia at room temperature, the snails were transferred to Petri dishes containing the maintenance medium and maintained at room temperature (24-27 °C ). Thirty days post-infection, each exposed snail was deposited in a 100-ml glass beaker containing a piece of yellow wax paper and water. The beaker was put in an ice-water bath to stimulate the emergence of cercariae; the latter encysted on the yellow wax paper as described by Durie (1955). The metacercariae were counted. The snails which did not shed any cercariae or which were dead were dissected to determine if they harboured the larval stage of the trematode.
Infection of sheep To confirm the identity of the isolated metacercariae, they were stored for 40 days, pooled and used for infecting Pelibuey sheep which were reared, housed and maintained in paramphistome-free conditions. Ten 2-year-old sheep were divided into five groups of two animals. Each group was infected orally with 50, 100, 500, 1000 or 5000 metacercariae. One sheep from each group was slaughtered at 70 days and the other 180 days post-infection. At necropsy, the paramphistomes were recovered from the rumen and counted. A representative number of recovered worms was relaxed, flattened between two slides and fixed in normal saline containing 10% formalin. They were stained by Gomori's stain and mounted for specific identification according to Dinnik (1961) and Kotrla and Prokovic (1973). RESULTS Of the five lymnaeid species exposed to P. cervi miracidia, only three species, L. cubensis, L. humilis and L. palustris, picked up the infection and L. bulimoides and L. columella proved refractory. A larger proportion of the exposed L. palustris was infected and shed cercariae than L. cubensis and L. humilis (Table 1 ). An infection dose of 6 miracidia produced 100% infection in L. palustris, whereas an exposure dose of > 12 miracidia was necessary to produce a 50% infection rate in the other two snail species; the rate of infection in these two species never reached 100% (Table 1 ). The mortality of the exposed L. palustris increased with an increase in the exposure dose with miracidia. It was higher in the case of L. palustris than in L. hurnilis and L. cubensis although, in relative terms, the latter two species showed a similar rate of mortality to the former in relation to their rate of infection. In the three species of Lymnaea successfully infected with P. cervi and surviving for 35 days post-infection and shedding cercariae, the number of cercariae discharged was inversely related to the miracidial exposure dose; the
160
L. CASTRO-TREJO ET AL.
'FABLE 1
Susceptibility, mortality and cercarial emergence from L. cubensis, L. humilis and L. palustris exposed to different numbers of Paramphistomum miracidia 1 Miracidia dose
2 4 6 8 10 12 14 16 18 20
Control
L. cubensis
L. humilis
L. palustris
No.
No.
No.
No.
No.
No.
No.
No.
infected
dead
shed
infected
dead
shed
infected
dead
25 30 41 40 45 57 59 60 60 60 0
1 4 4 5 14 33 58 60 60 60 0
3 4 2 1 0 0 0 -
13 25 33 35 35 39 47 57 53 58 0
8 1 1 4 14 27 34 53 49 56 0
1 1 1 1 1 0 0 0 0 0 0
89 98 100 100 100 100 100 100 100 100 0
0 2 2 24 79 99 92 95 100 100 0
0
No. shed 65 55 30 9 1 0 0 0
0
1Eleven groups of 100 snails each.
snail infected with two miracidia shed a high number of cercariae and the number decreased with an increase in the exposure dose. The sheep in all five groups, infected orally with different doses of metacercariae, showed the presence of P. cervi in their rumens. The sheep slaughtered at 70 days post-infection showed only juvenile parasites, whereas at 180 days post-infection, both juvenile and adult parasites were recovered, though the relative proportion of adults were less than juveniles. In both the instances, the number of worms recovered was dependent on the level of infection dose. DISCUSSION
AND CONCLUSION
The epidemiology of paramphistomiasis has not been studied exhaustively in Mexico. The snails of the genus Lymnaea spp., which serve as the intermediate host of Fasciola hepatica, are widely distributed in different regions of Mexico where cattle and sheep are raised (Castro-Trejo et al., 1983). This study shows that L. cubensis, L. humilis and L. palustris may act as intermediate hosts of P. cervi in Mexico. L. bulimoides and L. columeUa, were earlier reported to be intermediate hosts of P. cervi (Olsen, 1974), but in this study, both these species were refractory to experimental infections. L. palustris seems to maintain the parasite in nature and may help propagate the infection in Mexican ruminants. When snails were infected with fewer miracidia, they shed large numbers of cercariae; this has epizootiological implications. The other two lymnaeid species, L. cubensis and L. humilis could also be infected with P. cervi under laboratory conditions and, from the epizootiolog-
INFECTIONOFLYMNAEIDSWITHPARAMPHISTOMUMCERVI
161
ical point of view, these may appear to be less effective transmitters of the trematode. The maturation of the metacercariae to immature and mature stages of adult P. cervi in sheep confirmed that the cercariae shed by the infected snails, were viable and that the molluscan phase of the life cycle of the parasite was successfully completed. Traditionally, the introduction of Pelibuey sheep to cattle farms is a common practice in the tropical regions of Mexico. This situation offers an opportunity for the introduction and propagation of parasites in cattle, especially where the Lymnaea species are abundant. In conclusion, three species of Lyrenaea snails have been found to act as intermediate hosts of P. cervi under experimental conditions. To discover more about the epizootiology of paramphistomiasis, it is necessary to investigate further the relative distribution of various species of Lymnaea as well as the prevalence of snails infected with P. cervi in Mexico. REFERENCES Boray, J.C., 1959. Studies on intestinal amphistomosis in cattle. Aust. Vet. J., 35:282 287. Campos, R.R., Escutia, S.I. and Herrera, R.D., 1983. Estudio epizootiologico de algunas parasitosis internas de bovinos en el Istmo de Tehuantepec. In: Una ddcada de investigacidn en el Departamento de Parasitologia ( 1972-1982 ). Instituto Nacional de Investigaciones Pecuarias, SARH, Mexico, 64 pp. Castro-Trejo, L., Mata, E., Gonzalez, O. and Garcfa-Vasquez, Z., 1983. Estudio integrado de la Fasciolasis Bovina en el Estado de Durango. Memorias. Reunion de Investigaciones Pecuarias, SARH, M~xico, pp. 254-257. Castro-Trejo, L. and Casildo-Nieto, J., 1984. Vectores de la distomatosis hepatica an M~xico. IV. Congreso Nacional de Parasitologia Minatitlan Ver. M6xico, 58 pp. De la 0., J.R., Acevedo, A.H. and Quintero, M., 1983. Frecuencia y determinaci6n de trematodos de la fhmilia Paramphistomatidae (Fischoeder, 1901 ) de bovinos sacrificados en el frfgorifico y empacadora de Tabasco, S.A., Villahermosa Tabasco. Mere. Reun. An. Parasitol. Vet., Mexico, 16 pp. Dinnik, J.A., 1961. Paramphistomum phillerouxi (Trematoda): Paramphistomatidae and its development in Bulinus forskalli. J. Helminthol., 35: 69-90. Durie, P.H., 1955. A technique for collection of large number of paramphistome (Trematoda) metacercariae. Aust. J. Agric. Res., 6: 200-202. Horak, T.G., 1967. Host-parasite relationship of Paramphistomum microbothrium Fischoeder, 1901 in experimentally infected ruminants with particular reference to sheep. Ondersterpoor. J. Vet. Res., 34: 451-540. Kendall, S.B. and Parffit, J.W., 1959. Studies on the susceptibility of some species of Lymnaea to infection with Fasciola gigantica and F. hepatica. Ann. Trop, Med. Parasitol., 53: 220-227. Kotrla, B. and Prokovic, J., 1973. Paramphistomiasis of cattle in Cuba. Acta Vet., 42:35 44. Olsen, O.W., 1974. Animal Parasites, Their Life Cycles and Ecology. 3rd edn. University Press, pp. 323-342. Quiroz, H. and Ochoa, R., 1972. Presencia de Paramphistomum cervi (Schrank 1790 ), en un ovino de raza Tabasco 6 Pelibuey en M6xico. Tec. Pec. Mex., 21: 59. Soulsby, E.J., 1974. Helminths, Arthropods and Protozoa of Domestic Animals. Williams & Wilkins, Baltimore, MD, 64 pp. Taylor, E.L. and Mozley, A., 1948. A culture method for Lymnaea truncatula. Nature (London), 161: 594.
