V_eterinaryParasitology, 40 ( 1991 ) 119-126 Elsevier Science Publishers B.V., Amsterdam
119
Experimental Trichinella spiralis infection in sheep O. Tomagovi6ovfi, J. Corba, K. Havasiovfi, M. Rybog and A. Stefan6ikov~i Helminthological Institute of the Slovak Academy of Sciences, Dukelsky;ch hrdinov 3, 04001 Ko$ice, Czechoslovakia (Accepted 8 March 1991 )
ABSTRACT Toma~ovi~ovfi, O., t~orba, J., Havasiov~i, K., Rybog, M. and Stefan~ikov~i, A., 1991. Experimental Trichinella spiralis infection in sheep. Vet. ParasitoL, 40:119-126. During susceptibility studies of non-specific hosts, three merino sheep were infected with 3000, 5000 or 7000 Trichinella spiralis larvae by gavage. Clinical, physiological and serological parameters were assessed during the experiment. On the 152rid day p.i., animals were necropsied and, using artificial digestion methods, numbers of Trichinella larvae in muscle tissues were determined. The most infected parts were masseters with 3122 larvae g - l muscle, 5526 larvae g-1 muscle and 4058 larvae g-~ muscle and diaphragms with 2778 larvae g- ~muscle, 2725 larvae g- ~muscle and 2320 larvae g muscle, for the 3000, 5000 and 7000 infection levels, respectively. A positive correlation between infective rate and circulating antibodies was observed using ELISA and latex agglutination (LA) test methods. Trichinella larvae from sheep applied by gavage to ICR mice developed to the muscle stage. No significant changes were found in the clinical and physiological parameters of infected animals. Our results confirm the high susceptibility of merino sheep to T. spiralis infection.
INTRODUCTION
Susceptibility of non-specific hosts to Trichinella spiralis has been reported by several authors. Infection in horses and subsequent outbreaks of human trichinellosis was reported by Bellani et al. (1976) and others. Experimental T. spiralis infection in horses was described by Khalina et al. (1988) and Soule et al. (1989), in cattle by Billings ( 1883, cited in Campbell, 1983), in goats by Beck (1970) and in camels by Bommer et al. (1980). In reindeer, a natural T. spiralis infection was reported by Bessonov (1981 ). Smith and Snowdon (1989) found that T. spiralis became established in sheep when infective larvae were administered via gavage or by the feeding of sheep on infected muscle. These authors used high infective doses (22 500-100 000 larvae per animal). Our aim was to study susceptibility of sheep infected with lower doses of T. spiralis (3000-7000 larvae per animal) as well as some 0304-4017/91/$03.50
© 1991 Elsevier Science Publishers B.V. All rights reserved.
120
o. TOMASOVI(~OVAETAL.
physiological, serological, biochemical and clinical parameters during develo p m e n t of trichinellosis. MATERIALSAND METHODS T. spiralis source The Trichinella spiralis isolate used in our experiment originated from Vladivostok, USSR (supplied by Dr. Britov who isolated it from a wild boar) and which since 1982 has been passaged in specific pathogen free/ICR mice at the Helminthological Institute of the Slovak Academy of Sciences.
Experimental design and sampling Infective T. spiralis larvae were administered to three lambs by gavage after 24 h starvation as follows: (a) lamb No. 1, weighing 28 kg, received 3000 larvae, i.e. 107 kg -~ body weight (BW); (b) lamb No. 2, weighing 33.8 kg, received 5000 larvae, i.e. 148 kg -~ BW; (c) lamb No. 3, weighing 33 kg, received 7000 T. spiralis larvae, i.e. 212 kg- 1 BW.
Experimental animals Three Slovak merino lambs, 7-8 months old, were transferred from the Institute farm to the Institute 7 days before infection. They were kept separately in stalls with concrete floors which were cleaned daily and were fed on hay and concentrates.
Clinical observations At 7-day intervals, rectal temperature, white blood cell counts and egg per gram using the McMaster faecal examination m e t h o d were observed.
Serological test methods Antigen from muscle T. spiralis larvae was prepared by ultrasonic disintegration using an Ultrasonic Dismembrator 300 W Dynatech, followed by extraction in phosphate buffered saline at pH 7.2-7.4 for 24 h at 4°C. The homogenate was centrifuged for 15 min at 1250 g. The antigen obtained was standardised according to protein content using the method of Lowry et al. (1951). Blood samples for latex agglutination (LA) and ELISA tests were taken from v. jugularis on days 0, 11, 19, 35, 50, 62, 72, 92, 106, 119, 138 and 152 after infection.
EXPERIMENTAL TRICHINELLA SP1RALIS INFECTION IN SHEEP
121
Biochemical test methods Activity of alanine aminotransferase (AIaAT) and aspartate aminotransferase (AspAT) in blood serum was determined at the same intervals using a Biotest kit (Lachema, Brno, Czechoslovakia).
Autopsy procedure Animals were slaughtered at the local abattoir 152 days after infection. Samples weighing 50 g were taken from nine muscles (masseter, diaphragm, tongue, m.longissimus dorsi, m.deltoides, m.extensor capriradialis, m.biceps femoris, m.fibularis longis and ram. intercostales) according to Popesko ( 1968 ). Samples were digested in pepsin and HC1 solution according to Spaldonov~ et al. (1980) and larvae counted in an electronic counter according to the method of Velebn~ et al. ( 1979 ). These larvae were also used for infectivity trials in 30 SPF/ICR mice. Each mouse received 200 muscle larvae and on the 40th day p.i., animals were necropsied and processed according to the method ofSpaldonov~ et al. (1980) (Figs. 1 and 2).
Fig. 1. Trichinella spiralis on the 152nd day p.i. in the femoral muscle of sheep infected with 5000 larvae.
122
O. TOMASOVICOV& ET AL.
Fig. 2. Trichinellaspiralis on the 40th day p.i. in muscle of mice infected with 200 larvae obtained from infected sheep muscle. RESULTS
T. spiralis findings The distribution of muscle T. spiralis larvae in nine different parts of the musculature are shown in Table 1. The highest intensity of infection was found in all animals in masseter, diaphragm and tongue. A m a x i m u m finding of 5526 larvae g-~ muscle was recorded in the masseter of sheep No. 2 infected with 5000 T. spiralis larvae. All 30 mice infected with muscle larvae obtained from slaughtered sheep became infected with an infection rate comparable to trichinella establishment in control animals.
Clinical observations Clinical signs of illness were not observed during the course of the experiment. Body temperature and white blood counts were also within physiological range.
2778 2725 2320
1742 1866 2318
911 701 1227
845 1111 1146
998 1097 1008
1399 380 2259
770 556 870
803 1021 1138
3122 5526 4058
1 2 3
3000 5000 7000
M. biceps M. fibularis M. intercostales femoris longis
Sheep Infective dose Muscle No. (no. of larvae per animal) Masseter Diaphragm Tongue M. longis. M. deltoides M. extensor dorsi carpiradialis
Distribution of Trichinella spiralis larvae in muscles of infected sheep ( Trichinella larvae g ' muscle )
TABLE 1
1485_+ 841 1665_+1524 1816_+ 973
Average
~:
124
o. TOMA~OVICOV,~ETAL
600 t
500
t r @ s
400
/
\
i
300 200 "100
, ,) :3
0
77
! )d
I,~S
,c av's p, i,
Fig. 3. Dynamics of circulating antibodies (LA test) in infected sheep.
2,30C
~50C
) 'S,(
bOC
,,
Fig. 4. Dynamics of circulating antibodies (EL1SA) in infected sheep.
EXPERIMENTAL TRICHINELLA SPII~4LIS INFECTION IN SHEEP
125
Serological observations Antitrichinellous antibodies were found by LA test, using somatic larval antigen of T. spiralis on the 1 lth day in 1 : 8 and 1 : 32 titres. Maximum titres of 1 : 256 were observed on the 35th day p.i. and on the 50th day declined to 1:64 and remained at this value until the termination of the experiment (Fig. 3). Using the ELISA m e t h o d with somatic larval antigen, antibodies to T. spiralis were first found on the 1 lth day p.i. in titres of 1 : 50. Maximal titres of 1 : 1600 were observed in sheep No. 3 (infected with 7000 T. spiralis larvae) on the 35th day p.i. A delay of 8 days in the appearance of serum antibodies was detected in sheep No. 1, infected with 3000 T. spiralis larvae (Fig. 4).
Biochemical observations Activity of AlaAT and AspAT as indicators of muscle damage reached a m a x i m u m on the 19th day p.i. but in all animals remained at physiological levels during the whole experiment. DISCUSSION
Since 1976, several outbreaks of h u m a n trichinellosis have been attributed to consumption of infected horse meat. No outbreaks have been linked to ingestion of sheep meat. Recently, Smith and Snowdon (1989) successfully infected adult sheep with T. spiralis using high doses of muscle larvae (22 500100 000 larvae per animal). Trichinella spiralis nativa had a low infectivity for sheep, although light infection in some animals was established. The numbers of muscle T. spiralis larvae found in their experiments were significantly lower than those found in the present study. The highest infection rate found by these authors was 2738 larvae g- ~ muscle in the diaphragm of sheep infected with 80 000 larvae. In this experiment, the highest infection rate was 5526 larvae g-~ muscle in the masseter of a lamb infected with only 5000 T. spiralis larvae. Differences in susceptibility might be attributed to breed and age of experimental animals and the length of infections before slaughter. It is possible that between 33 and 56 days p.i. some muscle larvae were not encapsulated and thus not revealed (or were destroyed) by digestion. Positive serological findings in both the LA and ELISA tests confirmed the immunological response of infected animals during the development of the infection. The rapid production of antibodies and titres was proportional to infective doses and the number of muscle larvae at necropsy. Our results revealed a higher sensitivity of the ELISA test for the detection of sheep trichinellosis. It is noteworthy that muscle larvae obtained from sheep remained infective for rodents. It might be assumed that, under some circumstances, small ruminants can play an epidemiological role in spreading trichinellosis.
126
o. TOMASOVICOVAET AL.
REFERENCES
Beck, J.W., 1970. Trichinosis in domesticated and experimental animals. In: S.E. Gould (Editor), Trichinosis in Man and Animals, Charles C. Thomas, Springfield, IL, pp. 61-80. Bellani, I_., Mantovani, A., Pampigliane, L.S. and Filippini, J., 1976. Observations on an outbreak of human trichinellosis in northern Italy. In: Ch.W. Kim and Z.S. Pawlowski (Editors), Proceedings of the 4th International Conference on Trichinellosis, 26-28 August 1976. University Press of New England, pp. 48-52. Bessonov, A.S., 1981. Changes in the epizootic and epidemic situation of trichinellosis in the USSR. In: Ch.W. Kim, E.J. Ruitenberg and J.S. Teppema (Editors), Proceedings of the 5th International Conference on Trichinellosis, 1-5 September 1981. Reedbooks, Chertsey, UK, pp. 365-368. Billings, F.S., 1883. The relation of animal diseases to the public health and their prevention. Appleton, New York, pp. 2-41, 1884. Cited in: W.C. Campbell. Trichinella and Trichinosis, 1983. Plenum Press, New York and London, pp. 425-444. Bommer, W., Kaiser, H., Mergeziava, H. and Pottkamer, G., 1980. An outbreak of trichinellosis in northern Germany caused by imported air-dried camel meat. Abstracts of the 10th International Congress on Tropical Medicine and Malaria, Manila, 1980, p. 156. Cited in: Campbell, W.C. (Editor), Trichinella and Trichinosis, 1983, Plenum Press, New York and London, pp. 367-401. Campbell, W.C., 1983. Epidemiology. 1. Modes of transmission. In: W.C. Campbell (Editor), Trichinella and Trichinosis. Plenum Press, New York, 425-444, Khalina, R., Bessonov, A.S. and Belousov, M.N., 1988. Experimental infection of horses with 7Wchinella spiralis. Localization of larvae in muscles. In: Proceedings of tile 6th International Conference of Trichinellosis, 2-6 October 1988, Alicante, Programme and Abstracts. Graficus DIAZ, Alicante, Spain, p. 36. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Popesko, P., 1968. Atlas of Topographic Anatomy of Domesticated Animals. Parts I, II and Ill. SVPL, Bratislava. Smith, H.I. and Snowdon, K.E., 1989. Experimental trichinosis in sheep. Can. J. Vet. Res., 53: 112-114. Soule, C., Dupouy-Camet, J., Georges, P., Ancelle, T., Gillet, J., Vaissaire, J., Delvigne, A. and Plateau, E., 1989. Experimental trichinellosis in horses: Biological and parasitological evaluation. Vet. Parasit., 31: 19-36. Spaldonovfi, R., Velebn~, S., Tomagovi6ov& O. and Corba, J., 1980. Study of the efficacy of newly-synthesised drugs of Czechoslovak origin on model helminths. Final Rep. Helminth. Inst. Slovak Acad. Sci., Kogice, pp. 1-138. Velebn~, S., Spaldonov& R. and (~orba, J., 1979. Electronic counting of Trichinella spirahs larvae. Helminthologia, 16:199-206.
119
Experimental Trichinella spiralis infection in sheep O. Tomagovi6ovfi, J. Corba, K. Havasiovfi, M. Rybog and A. Stefan6ikov~i Helminthological Institute of the Slovak Academy of Sciences, Dukelsky;ch hrdinov 3, 04001 Ko$ice, Czechoslovakia (Accepted 8 March 1991 )
ABSTRACT Toma~ovi~ovfi, O., t~orba, J., Havasiov~i, K., Rybog, M. and Stefan~ikov~i, A., 1991. Experimental Trichinella spiralis infection in sheep. Vet. ParasitoL, 40:119-126. During susceptibility studies of non-specific hosts, three merino sheep were infected with 3000, 5000 or 7000 Trichinella spiralis larvae by gavage. Clinical, physiological and serological parameters were assessed during the experiment. On the 152rid day p.i., animals were necropsied and, using artificial digestion methods, numbers of Trichinella larvae in muscle tissues were determined. The most infected parts were masseters with 3122 larvae g - l muscle, 5526 larvae g-1 muscle and 4058 larvae g-~ muscle and diaphragms with 2778 larvae g- ~muscle, 2725 larvae g- ~muscle and 2320 larvae g muscle, for the 3000, 5000 and 7000 infection levels, respectively. A positive correlation between infective rate and circulating antibodies was observed using ELISA and latex agglutination (LA) test methods. Trichinella larvae from sheep applied by gavage to ICR mice developed to the muscle stage. No significant changes were found in the clinical and physiological parameters of infected animals. Our results confirm the high susceptibility of merino sheep to T. spiralis infection.
INTRODUCTION
Susceptibility of non-specific hosts to Trichinella spiralis has been reported by several authors. Infection in horses and subsequent outbreaks of human trichinellosis was reported by Bellani et al. (1976) and others. Experimental T. spiralis infection in horses was described by Khalina et al. (1988) and Soule et al. (1989), in cattle by Billings ( 1883, cited in Campbell, 1983), in goats by Beck (1970) and in camels by Bommer et al. (1980). In reindeer, a natural T. spiralis infection was reported by Bessonov (1981 ). Smith and Snowdon (1989) found that T. spiralis became established in sheep when infective larvae were administered via gavage or by the feeding of sheep on infected muscle. These authors used high infective doses (22 500-100 000 larvae per animal). Our aim was to study susceptibility of sheep infected with lower doses of T. spiralis (3000-7000 larvae per animal) as well as some 0304-4017/91/$03.50
© 1991 Elsevier Science Publishers B.V. All rights reserved.
120
o. TOMASOVI(~OVAETAL.
physiological, serological, biochemical and clinical parameters during develo p m e n t of trichinellosis. MATERIALSAND METHODS T. spiralis source The Trichinella spiralis isolate used in our experiment originated from Vladivostok, USSR (supplied by Dr. Britov who isolated it from a wild boar) and which since 1982 has been passaged in specific pathogen free/ICR mice at the Helminthological Institute of the Slovak Academy of Sciences.
Experimental design and sampling Infective T. spiralis larvae were administered to three lambs by gavage after 24 h starvation as follows: (a) lamb No. 1, weighing 28 kg, received 3000 larvae, i.e. 107 kg -~ body weight (BW); (b) lamb No. 2, weighing 33.8 kg, received 5000 larvae, i.e. 148 kg -~ BW; (c) lamb No. 3, weighing 33 kg, received 7000 T. spiralis larvae, i.e. 212 kg- 1 BW.
Experimental animals Three Slovak merino lambs, 7-8 months old, were transferred from the Institute farm to the Institute 7 days before infection. They were kept separately in stalls with concrete floors which were cleaned daily and were fed on hay and concentrates.
Clinical observations At 7-day intervals, rectal temperature, white blood cell counts and egg per gram using the McMaster faecal examination m e t h o d were observed.
Serological test methods Antigen from muscle T. spiralis larvae was prepared by ultrasonic disintegration using an Ultrasonic Dismembrator 300 W Dynatech, followed by extraction in phosphate buffered saline at pH 7.2-7.4 for 24 h at 4°C. The homogenate was centrifuged for 15 min at 1250 g. The antigen obtained was standardised according to protein content using the method of Lowry et al. (1951). Blood samples for latex agglutination (LA) and ELISA tests were taken from v. jugularis on days 0, 11, 19, 35, 50, 62, 72, 92, 106, 119, 138 and 152 after infection.
EXPERIMENTAL TRICHINELLA SP1RALIS INFECTION IN SHEEP
121
Biochemical test methods Activity of alanine aminotransferase (AIaAT) and aspartate aminotransferase (AspAT) in blood serum was determined at the same intervals using a Biotest kit (Lachema, Brno, Czechoslovakia).
Autopsy procedure Animals were slaughtered at the local abattoir 152 days after infection. Samples weighing 50 g were taken from nine muscles (masseter, diaphragm, tongue, m.longissimus dorsi, m.deltoides, m.extensor capriradialis, m.biceps femoris, m.fibularis longis and ram. intercostales) according to Popesko ( 1968 ). Samples were digested in pepsin and HC1 solution according to Spaldonov~ et al. (1980) and larvae counted in an electronic counter according to the method of Velebn~ et al. ( 1979 ). These larvae were also used for infectivity trials in 30 SPF/ICR mice. Each mouse received 200 muscle larvae and on the 40th day p.i., animals were necropsied and processed according to the method ofSpaldonov~ et al. (1980) (Figs. 1 and 2).
Fig. 1. Trichinella spiralis on the 152nd day p.i. in the femoral muscle of sheep infected with 5000 larvae.
122
O. TOMASOVICOV& ET AL.
Fig. 2. Trichinellaspiralis on the 40th day p.i. in muscle of mice infected with 200 larvae obtained from infected sheep muscle. RESULTS
T. spiralis findings The distribution of muscle T. spiralis larvae in nine different parts of the musculature are shown in Table 1. The highest intensity of infection was found in all animals in masseter, diaphragm and tongue. A m a x i m u m finding of 5526 larvae g-~ muscle was recorded in the masseter of sheep No. 2 infected with 5000 T. spiralis larvae. All 30 mice infected with muscle larvae obtained from slaughtered sheep became infected with an infection rate comparable to trichinella establishment in control animals.
Clinical observations Clinical signs of illness were not observed during the course of the experiment. Body temperature and white blood counts were also within physiological range.
2778 2725 2320
1742 1866 2318
911 701 1227
845 1111 1146
998 1097 1008
1399 380 2259
770 556 870
803 1021 1138
3122 5526 4058
1 2 3
3000 5000 7000
M. biceps M. fibularis M. intercostales femoris longis
Sheep Infective dose Muscle No. (no. of larvae per animal) Masseter Diaphragm Tongue M. longis. M. deltoides M. extensor dorsi carpiradialis
Distribution of Trichinella spiralis larvae in muscles of infected sheep ( Trichinella larvae g ' muscle )
TABLE 1
1485_+ 841 1665_+1524 1816_+ 973
Average
~:
124
o. TOMA~OVICOV,~ETAL
600 t
500
t r @ s
400
/
\
i
300 200 "100
, ,) :3
0
77
! )d
I,~S
,c av's p, i,
Fig. 3. Dynamics of circulating antibodies (LA test) in infected sheep.
2,30C
~50C
) 'S,(
bOC
,,
Fig. 4. Dynamics of circulating antibodies (EL1SA) in infected sheep.
EXPERIMENTAL TRICHINELLA SPII~4LIS INFECTION IN SHEEP
125
Serological observations Antitrichinellous antibodies were found by LA test, using somatic larval antigen of T. spiralis on the 1 lth day in 1 : 8 and 1 : 32 titres. Maximum titres of 1 : 256 were observed on the 35th day p.i. and on the 50th day declined to 1:64 and remained at this value until the termination of the experiment (Fig. 3). Using the ELISA m e t h o d with somatic larval antigen, antibodies to T. spiralis were first found on the 1 lth day p.i. in titres of 1 : 50. Maximal titres of 1 : 1600 were observed in sheep No. 3 (infected with 7000 T. spiralis larvae) on the 35th day p.i. A delay of 8 days in the appearance of serum antibodies was detected in sheep No. 1, infected with 3000 T. spiralis larvae (Fig. 4).
Biochemical observations Activity of AlaAT and AspAT as indicators of muscle damage reached a m a x i m u m on the 19th day p.i. but in all animals remained at physiological levels during the whole experiment. DISCUSSION
Since 1976, several outbreaks of h u m a n trichinellosis have been attributed to consumption of infected horse meat. No outbreaks have been linked to ingestion of sheep meat. Recently, Smith and Snowdon (1989) successfully infected adult sheep with T. spiralis using high doses of muscle larvae (22 500100 000 larvae per animal). Trichinella spiralis nativa had a low infectivity for sheep, although light infection in some animals was established. The numbers of muscle T. spiralis larvae found in their experiments were significantly lower than those found in the present study. The highest infection rate found by these authors was 2738 larvae g- ~ muscle in the diaphragm of sheep infected with 80 000 larvae. In this experiment, the highest infection rate was 5526 larvae g-~ muscle in the masseter of a lamb infected with only 5000 T. spiralis larvae. Differences in susceptibility might be attributed to breed and age of experimental animals and the length of infections before slaughter. It is possible that between 33 and 56 days p.i. some muscle larvae were not encapsulated and thus not revealed (or were destroyed) by digestion. Positive serological findings in both the LA and ELISA tests confirmed the immunological response of infected animals during the development of the infection. The rapid production of antibodies and titres was proportional to infective doses and the number of muscle larvae at necropsy. Our results revealed a higher sensitivity of the ELISA test for the detection of sheep trichinellosis. It is noteworthy that muscle larvae obtained from sheep remained infective for rodents. It might be assumed that, under some circumstances, small ruminants can play an epidemiological role in spreading trichinellosis.
126
o. TOMASOVICOVAET AL.
REFERENCES
Beck, J.W., 1970. Trichinosis in domesticated and experimental animals. In: S.E. Gould (Editor), Trichinosis in Man and Animals, Charles C. Thomas, Springfield, IL, pp. 61-80. Bellani, I_., Mantovani, A., Pampigliane, L.S. and Filippini, J., 1976. Observations on an outbreak of human trichinellosis in northern Italy. In: Ch.W. Kim and Z.S. Pawlowski (Editors), Proceedings of the 4th International Conference on Trichinellosis, 26-28 August 1976. University Press of New England, pp. 48-52. Bessonov, A.S., 1981. Changes in the epizootic and epidemic situation of trichinellosis in the USSR. In: Ch.W. Kim, E.J. Ruitenberg and J.S. Teppema (Editors), Proceedings of the 5th International Conference on Trichinellosis, 1-5 September 1981. Reedbooks, Chertsey, UK, pp. 365-368. Billings, F.S., 1883. The relation of animal diseases to the public health and their prevention. Appleton, New York, pp. 2-41, 1884. Cited in: W.C. Campbell. Trichinella and Trichinosis, 1983. Plenum Press, New York and London, pp. 425-444. Bommer, W., Kaiser, H., Mergeziava, H. and Pottkamer, G., 1980. An outbreak of trichinellosis in northern Germany caused by imported air-dried camel meat. Abstracts of the 10th International Congress on Tropical Medicine and Malaria, Manila, 1980, p. 156. Cited in: Campbell, W.C. (Editor), Trichinella and Trichinosis, 1983, Plenum Press, New York and London, pp. 367-401. Campbell, W.C., 1983. Epidemiology. 1. Modes of transmission. In: W.C. Campbell (Editor), Trichinella and Trichinosis. Plenum Press, New York, 425-444, Khalina, R., Bessonov, A.S. and Belousov, M.N., 1988. Experimental infection of horses with 7Wchinella spiralis. Localization of larvae in muscles. In: Proceedings of tile 6th International Conference of Trichinellosis, 2-6 October 1988, Alicante, Programme and Abstracts. Graficus DIAZ, Alicante, Spain, p. 36. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Popesko, P., 1968. Atlas of Topographic Anatomy of Domesticated Animals. Parts I, II and Ill. SVPL, Bratislava. Smith, H.I. and Snowdon, K.E., 1989. Experimental trichinosis in sheep. Can. J. Vet. Res., 53: 112-114. Soule, C., Dupouy-Camet, J., Georges, P., Ancelle, T., Gillet, J., Vaissaire, J., Delvigne, A. and Plateau, E., 1989. Experimental trichinellosis in horses: Biological and parasitological evaluation. Vet. Parasit., 31: 19-36. Spaldonovfi, R., Velebn~, S., Tomagovi6ov& O. and Corba, J., 1980. Study of the efficacy of newly-synthesised drugs of Czechoslovak origin on model helminths. Final Rep. Helminth. Inst. Slovak Acad. Sci., Kogice, pp. 1-138. Velebn~, S., Spaldonov& R. and (~orba, J., 1979. Electronic counting of Trichinella spirahs larvae. Helminthologia, 16:199-206.
