Muscular Changes in Venezuelan Wild Horses Naturally Infected with Trypanosoma evansi

J. Comp. Path. 1994 Vol. ll0, 79-89

Muscular Changes in Venezuelan Wild Horses Naturally Infected with Trypanosoma evansi M.E. Quifiones Mateu, H. J. Finol, L.E. Sucre* and S. H. Torres~ Center of Electron Microscopy, Faculties of Science, * Veterinary Medicine and ~Medicine, Central University of Venezuela, Apartado 47114, Los Chaguaramos, Caracas 1041 A, Venezuela

Summary Skeletal muscle biopsy specimens were taken from 10 male horses naturally infected with Trypanosoma evansi and from 10 uninfccted males. An indirect fluorescent antibody test was used to provide a rapid and reliable indication of infection. Histological, histochcmical and transmission electron microscopical techniques were used to examine skeletal muscle. The ultrastructural features in muscle fibres were those usually seen in autoimmune disease, namely fibre and capillary necrosis and mononuclear cell infiltration, consisting of macrophagcs. Changes in fibre-type percentages did not occur in response to the parasite, but alterations in fibre size and capillary supply were found in the parasitized horses.

Introduction T h e Venezuelan wild horse is a direct descendent of horses brought in by the Spanish conquerors. Parasitic infection with Trypanosoma evansi is known in Central and South America as "derrengadera" and in Asia as "surra" (Hoare, 1972; M a h m o u d and Gray, 1980). This disease is manifested in horses by increased temperature, anaemia and weakness. This disease may develop over a few months or years, leading to death (Hoare, 1972; Losos, 1980; Mahmoud and Gray, 1980; H6rchner et al., 1983). The pathogenesis of the disease is still uncertain and the events which lead to death are almost unknown. Most studies suggest that alterations of the locomotor system are preceded by changes in the nervous system (Losos, 1980; Mahmoud and Gray, 1980), but there is no evidence concerning muscular damage in horses infected with T. evansi. This paper deals with the histochemistry and ultrastructure of equine skeletal muscle in animals which, although infected with this parasite, did not show parasites intramuscularly.

Materials and Methods HOTS&S

Twenty male adult horses from Hato E1 Frlo in Venezuela were used in this investigation. All were living in the same environment and were subjected to the same Correspondence to: M. E. Quifiones Mateu at his present address: Centro de Biologia Molecular "Severo Ochoa", Facuhad de Ciencias, Universldad Autdnoma de Madrid, 28049 Madrid, Espafla. 0021-9975/941010079+ 11 $08.00/0

© 1994 Academic Press Limited

80

M. E, Quifiones Mateu et al.

nutritional conditions on lowland pastures of the Venezuelan prairie. They were selected on the basis of a serological test (see below). Horses nos 1-10, aged 2-12 years, were uninfected (titre 10). Horses 11-20, aged 3-9 years, were infected with 7-. evansi (titres 20-300, mean 64).

Diagnosis Clinical diagnosis of the disease was made by an indirect fluorescent antibody test (IFAT) (Luckins et al., 1978). The test was considered positive w h e n the titres were > 10. Blood samples for the IFAT were taken f?om the external jugular vein.

Biopsy Samples All muscle samples were obtained percutaneously from the left middle gluteal muscle, 15 cm caudodorsal to the tuber coxae at a depth of 10 cm, by means of the technique described by Lindholm and Piehl (1974). Upon collection, the muscle sample was divided into two portions, one for histochemical analysis and the other for ultrastructural study. The portion for histochemical analysis was covered with talcum powder to prevent the formation of ice artefacts, and was frozen in liquid nitrogen ( - 196°C). The portion for electron microscopical observation was prepared with a minimum of delay.

Electron Microscopical Analysis Pieces of muscle, 2 mm in diameter, were fixed with glutaraldehyde 3 per cent and OsO 4 1 per cent (phosphate-buffered solutions (pH 7"8; 320 mOsm), dehydrated in increasing ethanol concentrations, and embedded in epon. Sections were cut with a diamond knife in a Porter-Blum MT2-B ultramicrotome and stained with uranyl acetate and lead citrate (for further details see Finol and Ogura, 1977). Sections were examined with a Hitachi H-500 transmission electron microscope (100 kV accelerating voltage).

Histochemical Analysis Transverse serial sections were cut on a cryostat-microtome at - 20°C and mounted on coverslips for staining. Sections (10gin) were examined for myosin adenosine triphosphatase after pre-incubation with both acid buffer (pH4.37, 4'6 and 4.8) (Brooke and Kaiser, 1970) and alkaline buffer (pH 10'3) (Padykula and Herman, 1955), for reduced nicotinamide dinucleotide diaforase (NADH-d) (Novikoff et al., 1961) and for ~-glycerophosphate dehydrogenase (~-GPDH) (Wattemberg and Leong, 1960). The percentage &fast and slow fibres was determined from the section stained for myosin ATPase activity at pH 4"37. From the section stained for NADH-d, the percentage of high and low oxidative fibres was determined. Combining the results obtained with these two stains, the percentage of slow oxidative (SO) fibres corresponded to that of low rnyosin ATPase fibres, and the percentage of fast oxidative glycolytic (FOG) fibres corresponded to that of the low oxidative fibres. The percentage &fast glycolytic (FG) fibres was then obtained from the formula of Snow and Guy (1980): % F G = 1 0 0 - (%SO + %FOG). The number of capillaries surrounding each fibre type was determined by the s-amylase-periodic acid-Schiff (PAS) technique (Andersen and Henriksson, 1977). Photomicrographs of each section were taken, and with an image analyser (Ladd Microcomputer Graphic Data Analyzer System) the following data were obtained: mean area for each muscle fibre type (SO, FG, and FOG); capillary (cap) density (no.

81

T. evansi-indnced C h a n g e s in E q u i n e M u s c l e 50 1

% FIBRE TYPE 40

30

20

10

0 ,~

80 Fig. l.

R3

FOG

Mean values of the percentage of total muscle fibre population of each type (SO, FG, FOG) in 20 Venezuelan wild horses; 10 non-parasitized ([21) and 10 parasitized (~1). There were no statistically significant differences (P> 0.05) between the two groups.

cap per mma); the number of capillaries surrounding individual fibres, and capillary index (no. cap per no. fibres); and the mean area/number of capillaries surrounding each muscle fibre type. Atrophy and hypertrophy factors were calculated for all combined fibres. When a specific area was identified, a cross-haired stylet attached to the image analyser was used to measure the minimal fibre diameter. This technique was used to determine the normal range of small or large fibres in the muscle as described for horse muscle by Andrews and Spurgeon (1986). Abnormal histological characteristics, including muscle fibre shape, were identified in muscle sections stained with haematoxylin and eosin.

Statistical Analysis A standard Student "t" test was used in comparing the percentage of fibre type populations, mean area for each muscle fibre type, density, number, index and the mean area/number of capillaries.

Results

Histochemical Profiles T h e classification of muscle fibre types was performed according to Snow and G u y (1980). SO fibres predominated in all animals (normal and infected) studied (Fig. 1). Similar results were obtained from muscle fibre oxidative and glycolytic metabolic potentials. Analysis of photomicrographs stained for o~amylase-PAS showed a decrease of mean muscle fibre diameter in one animal infected with T. evansi. Although only two control (nos 1 and 2) and two

82

M.E.

Quifiones

Mateu

e t al.

Table 1 M e a n a r e a s (/tin 2) a n d c a p i l l a r y v a r i a b l e s for e a c h f i b r e type o f f o u r V e n e z u e l a n w i l d h o r s e s Values J'ound in non-parasitized horses no,

parasitized horses no.

A4easuTwmenl

Fibre type

l

2

Il

12

Mean area

SO FG FOG

5221 5414 10200 325 2"28 3-75 3'6I) 3.67

2214 2653 3750 296 1-78 3'00 2-36 2'50

4229 5275 7624 353 1'77 2-94 2"67 1'67

1392 1503 2779

738 1 I24 1500

1438 I975 4565

Capillary density Capillary index N u m b e r of capillaries adjacent to each fibre type

SO FG FOG

5201 5154 8933 328 1.97 3"96 3.13 2'59

M e a n area/Number of capitlarles adjacent to each fibre type

SO FG FOG

1313 1646 3449

infected horses (nos 11 and 12) were analysed for fibre area and capillaries, a difference was found in respect of capillary index, and n u m b e r of capillaries next to each type of fibre (Table 1). Haematoxylin/eosin staining showed that the skeletal muscle of all infected animals presented c o m m o n characteristics, such as haemorragic areas, the presence of mononuclear cell infiltrates and changes of size and shape.

Ultrastructural Profiles Figure 2 shows a section from normal F O G fibre. Myofibres consisted of sarcomers with characteristic bands and lines. Like SO fibres, F O G fibres exhibited a b u n d a n t mitochondria and lipid droplets in subsarcolemmal and intermyofibrillar spaces, which were narrow a n d contained numerous glycogen [3 particles. These particles were also a b u n d a n t in F G fibres. M i n o r changes consisted of loss of contractile elements with increased intermyofibrillar spaces (Fig. 3). These were occupied by very electron-dense rnitochondria, numerous glycogen particles, vacuolized spaces and a few triadic elements. Lysosomes were represented by lipofuscin granules with altered m o r p h o l o g y (Fig. 4). Nuclei appeared pyknotic and clustered in atrophic areas (Fig. 5). More striking changes consisted of supercontraction of myofibrils and the presence of mitochondria with different degrees of swelling (Fig. 6). Areas of necrosis were also observed, being represented by contractile masses in close association with macrophages (Fig. 7). Capillaries without a l u m e n (compare Fig. 8 with Fig. 2) and even necrotic ones were found. There was no correlation between the severity of the muscle lesions and the I F A T test titre. Fig. 9 illustrates the change in size of muscle fibres in parasitized animals.

Fig. 2. Fig, 3,

Electron micrograph of section of normal skeletal muscle: (circle) A band; (arrow) M line; (arrow head) Z line; (C) capillary, × 12000. Skeletal muscle of infected horse: (square) wide intermyofibrillar space; (circle) electron-dense mitoehondrion; (asterisk) vacuolated space; (arrow) prominent triad. EM. × 24 000.

Fig. 4-. Oblique muscle fibre section: (arrows) lipofnscin granule. Covering membrane is not visible. EM. x 30 000, Fig. 5, Subsarcolemmal space in muscle fibre: (circle) pycknotic nucleus, EM. × I0 000.

Fig. 6. Fig. 7.

Section of contracted muscle fibre, In subsarcolemmal space: (square) swelling mitochondria, EM. x 12 000. Necrotic muscle fibre section. Between contractile masses, a macrophage. EM. x 18 000.

86

Fig. 8. Fig. 9.

M. E. Quifiones Mateu e t al.

Capillary (C) without lumen. EM. x 15 000. Staining with ~-amylase-PAS shows the alteration of size in the muscle fibres of a parasitized animal, x 198.

T. evansi-induced Changes in Equine Muscle

87

Discussion Muscle atrophy in infected equines is similar to that observed in denervation (A1-Amood et al., 1986), autoimmune disorders (Finol et al., 1988, 1990) and other skeletal muscle diseases. As in studies of several equine neuromuscular disorders (De Lahunta, 1978; Andrews et al., 1986), no selective atrophy of any particular fibre type was observed. Electron microscopy showed the typical alterations of denervated muscle, together with others not usually observed in neurogenic atrophy. Changes of neural origin have been previously described (Shafiq el al., 1967; Gonzfi,lez de Milo el al., 1988; Borg et al., 1989). They include myofibrillar loss and sarcotubular system changes. Changes not usually seen in denervation atrophy are fibre necrosis, capillary abnormalities and infiltration of macrophages. Muscle fibre necrosis is a myopathie change observed in many primary myopathies; capillary changes, which include necrosis, have been reported in a single study on denervation atrophy (Carpenter and Karpati, 1982), and mononuclear cell infiltration, characteristic of inflammatory myopathies, has not been described in neurogenic atrophy (Shaflq et al., 1967; Borg et al., 1989). Co-existence of these three changes has been found in rheumatoid myositis (Finol et al., 1988), a pathological condition associated with rheumatoid arthritis. Capillary abnormalities and mononuclear cell infiltration have been observed in lupus myositis (Finol et al., 1990), Graves-Basedow disease (Finol el al., 1986) and in the "muscular paraneoplasic phenomenon" (M~irquez et al., 1989). I n all these cases the infiltration was formed by different cells, including lymphocytes, macrophages and mast cells. In the present study only macrophages were observed. This study confirms the observations of H6rchner el al. (1983), which indicated that T. evansi did not invade skeletal muscle. Because of the lack of invasion, the muscle damage must have been produced by an indirect mechanism. Trypanosomiasis can produce changes in the immune system conducive to autoimmunity, as seen in cancer (Beutler and Cerami, 1987; Fayer et al., 1988). Parasites may produce damage in non-invaded tissues by mechanisms similar to those found in the paraneoptastic phenomenon (Finol el al., 1991; Tonino et al., 1991). This is the first report of an inflammatory myopathy in horses caused by a parasite which does not invade the muscle cell.

Acknowledgments We thank Dr F. Garcia for providing technical assistance and Drs A. Mgtrquez and J. A. Est& for reviewing the manuscript. This work was supported by a grant from CDCH of UCV, No. 03.10.2709/92. References

A1-Amood, W. S., Finol, H.J. and Lewis, D. M. (1986). Chronic stimulation modifies the isotonic shortening veIocity ofdenervated rat slow-twitch muscle. Proceedings of the Royal Society, 228, 43-58. Andersen, P. and Henriksson, J. (1977). Capillary supply of the quadriceps femoris muscle of man: adaptative response to exercise. Journal of Physiology, 270, 677-690.

88

M.E. Quifiones Mateu e t al.

Andrews, F. M. and Spurgeon, T. L. (1986). Histochemical staining characteristics of normal horse skeletal muscle. AmericanJournal of VeterinaryResearch,47, 1843-1852. Andrews, F.M., Spurgeon, T. L. and Reed, S.M. (1986). Histochemical changes in skeletal muscle &four male horses with neuromuscular disease. AmericanJournal of Veterinary Research, 47, 2078-2083. Beutler, B. and Cerami, A. (1987). Cachetin: more than a tumor necrosis factor. New England Journal of Medicine, 316, 379-385. Borg, K. B., Solders, G., Burg, J., Edstron, L. and Kristensson, K. (1989). Neurogenetic involvement in distal myopathy (Welander). Histochemicat and morphological observations on muscle and nerve biopsies. Journal of Neurological Science, 91, 53-70. Brooke, M.H. and Kaiser, K.K. (1970). Muscle fiber types: how many and what kind? Archives of Neurolo~, 23, 369-379. Carpenter, S. and Karpati, G. (1982). Necrosis of capillaries in denervation atrophy of human skeletal muscle. Muscle and Nerve, 5, 250-254. De Lahunta, A. (1978). Diagnosis of equine neurologic problems. Cornell Veterinarian,7, 122-131. Fayer, R., Andrews, C. and Dubey, P. (1988). Lysates of Sarcocystis cruzi bradyzoites stimulate raw 264"7 macrophages to produce tumor necrosis factor (cathetin). Journal of Parasitology, 74, 660-664. Finol, H.J., Montagnani, S., M~rquez, A., Montes de Oca, I. and Miiller, B. (1990). Ultrastructural pathology of skeletal muscle systemic lupus erythematosus. Journal of Rheumatology, 17, 210-219. Finol, H.J., Mriller, B., Montes de Oca and M~rquez, A. (1988). Ultrastructure of skeletal muscle in rheumatoid myositis. Journal of Rheumatology, 15, 552-555. Finol, H.J., Mfiller, B., Torres, S. H., Dominguez, J.J., Perdomo, P. and Montes de Oca, I. (1986). Ultrastructural abnormalities in muscular vessels of hyperthyroid patients. Acta Neuropathologica, 71, 64--69. Finol, I--I.J. and Ogura, M. (1977). Estudio sobre los tipos de fibras musculares esquel~ticas de la iguana. Acta Cientifica Venezolana, 28, 213-219. Finol, H.J., Rodrlguez, I., M~irquez, A., Montes de Oca, I. and Miiller, B. (1991). An electron microscopy study of neuromuscular alterations in paraneoplastic phenomenon. Abstracts, Societyfor Neuroscience, 17, 1611. Gonz/dez de Milo, N., Finol, I-t.J. and M~rquez, A. (1988). Ultrastructural study of reinnervation in a rat fast skeletal muscle. Acta Cient(fica Venezolana, 39, 257-262. Hoare, C. (1972). The Trypanosomes of Mammals. A Zoological Monograph. Blackwell Scientific Publications, Oxford, pp. 555-593. H6rchner, F., Sch6nefeld, A. and Wrist, B. (1983). Experimental infection of horses with Trypanosoma evansi. Parasitological and clinical results. Annals Society Belge Mddicine Tropicale, 63, 127-135. Lindholm, A. and Piehl, K. (1974). Fibre composition, enzyme activities and concentrations of metabolites and electrolytes in muscles of Standardbred horses. Acta VeterinariaScandinavica, 15, 287-309. Losos, G.J. (1980). Diseases caused by Trypanosomaevansi. A review. VeterinaryResearch Communications, 4, 165-180. Luckins, A. G., Gray, A. R. and Rae, P. (1978). Comparison of the diagnostic value of serum immunoglobulin levels, an enzyme-linked immunosorbent assay and a fluorescent antibody test in experimental infections with Trypanosoma evansi in rabbits. Annals of Tropical Medicine and Parasitology, 72, 429-441. Mahmoud, M. M. and Gray, A. R. (1980). Trypanosomiasis due to Trypanosoma evansi (Steel, 1985) Balbiani, 1888. A review of recent research. Tropical Animal Health and Production, 12, 35-47. M~rquez, I., Blanco, Y., Tonino, P., Finol, H.J., Prieto, J. and Sosa, L. (1989). Ultrastructural pathology of skeletal muscle in paraneoplastic syndrome. Abstract, Societyfor Neuroscience, 15, 1033.

2". e v a n s i - i n d u c e d

Changes in Equine Muscle

89

Novikoff, A.B., Shine, W. and Drucker, J. (1961). Mitochondrial localization of oxidation enzymes: staining results with two tetrazolium salts. Journal of Biophysics and Biochemistry Cytology, 9, 47-61. Padykula, H. A. and Herman, E. (1955). The specificity of the histochemical method for adenosine triphosphatase. Journal of Histochemistryand Cytochemistry,3, 170-195. Shafiq, S.A., Gorycki, M.A. and Milhorat, A.T. (1967). An electron microscopic study of regeneration and satellite cells in human muscle. Neurology, 17, 567-574. Snow, D. H. and Guy, P. S. (1980). Muscle fibre type composition of a number of limb muscles in different types of horse. Research in VeterinaryScience, 28, 137-144. Tonino, P., Finol, H.J., M~rquez, A. and Prieto, J. (1991). UItrastructural pathology of skeletal muscle in the paraneoplastic phenomenon. Journal of Experimental and Clinical Cancer Research, 10, 283-289. Wattemberg, L. W. and Leong, J. L. (1960). Effects of coenzyme Q 10 and medanione on succinate dehydrogenase activity as measured by tetrazolium salt reduction. Journal of Histochemistry and Cytochemistry, 8, 296-300.

~ Received, September l Oth, 1992] Accepted, October4th, 1993 _]