Infected Muscle Fibers Research Articles

G.P.78: Acute Myositis due to Sarcocystis nesbitii infection

Human muscle sarcocystosis is usually asymptomatic but can present monosymptomatically as focal muscle pain and/or swelling or rarely, with acute systemic symptoms in an outbreak. We describe features of acute myositis seen in an outbreak of Sarcocystis nesbitii infection in Malaysia in 2012. Infected patients presented acutely with fever and generalised myalgia, which in some included the face and jaw muscles. In about half the patients, myalgia was moderate to severe. There were 17 patients in whom myositis could be confirmed either by the presence of painful muscle swelling or hyperintensity on MRI STIR sequences suggesting muscle inflammation. Muscle swelling and inflammation on MRI were multifocal rather than diffuse and there was predominant involvement of the facial and masticatory muscles suggesting a possible predilection of the sarcocysts for these muscles. Muscle biopsy confirmed the diagnosis by demonstrating the presence of intracellular sarcocysts which was confirmed to be S. nesbitii by molecular methods. Inflammatory changes did not surround the infected muscle fibres but were seen focally with necrosis elsewhere within the muscle. Tissue eosinophilia was not prominent. Laboratory investigations revealed elevated serum creatine kinase and eosinophil count only in a third and half of the patients respectively. Even without specific treatment, muscle pain and swelling gradually subsided over a period of weeks to months. In summary, acute S. nesbitii myositis presented with fever and multifocal muscle pain and swelling. The multifocal nature of muscle inflammation was confirmed on MRI. The intracellular location of the sarcocysts does not elicit a direct immune response but sarcocysts may secrete chemical antigens that result in inflammation elsewhere.

Development ofTrichosomoides nasalis(Nematoda: Trichinelloidea) in the murid host: evidence for larval growth in striated muscle fibres

Trichosomoides nasalis (Trichinelloidea) is a parasite of Arvicanthis niloticus (Muridae) in Senegal. Female worms that harbour dwarf males in their uteri, occur in the epithelium of the nasal mucosa. Young laboratory-bred A. niloticus were either fed females containing larvated eggs or intraperitoneally injected with motile first-stage larvae recovered from female uteri. Both resulted in successful infection. Organs examined during rodent necropsy were blood and lymphatic circulatory systems (heart, large vessels, lymphnodes), lungs, liver, kidneys, thoracic and abdominal cavities, thoracic and abdominal muscular walls, diaphragm, tongue, and nasal mucosa. Development to adult nasal stages took three weeks. Recovery of newly hatched larvae from the peritoneal fluid at four-eight hours after oral infection suggests a direct passage from the stomach or intestinal wall to the musculature. However, dissemination through the blood, as observed with Trichinella spiralis, cannot be excluded even though newly hatched larvae of T. nasalis are twice as thick (15 μm). Developing larvae were found in histological sections of the striated muscle of the abdominal and thoracic walls, and larvae in fourth moult were dissected from these sites. Adult females were found in the deep nasal mucosa where mating occurred prior to worms settling in the nasal epithelium. The present study shows a remarkable similarity between T. nasalis and Trichinella species regarding muscle tropism, but the development of T. nasalis is not arrested at the late first-larval stage and does not induce transformation of infected fibres into nurse cells. T. nasalis seems a potential model to study molecular relations between trichinelloid larvae and infected muscle fibres.

Trichinella spiralis infection induces angiogenic factor thymosin β4 expression

Trichinella spiralis (T. spiralis) has been reported to up-regulate the expression of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. In order to analyze the induction of angiogenesis by T. spiralis, the expression patterns of angiogenesis-related proteins were investigated by immunohistochemical analysis. VEGF expression was induced in the infected muscles at an early stage of infection (10 days after infection) and diminished after 3 weeks. Thymosin β4, a major factor which induces VEGF, showed increased expression in muscle fibers 10 days after infection, and the expression remained high in the nurse cells for 6 weeks, when the formation of the nurse cell complex was completed. The hypoxia inducible factor (HIF)-1α showed a diffuse expression pattern around the infected muscle fibers and was strongly expressed in inflammatory cells but was not related to the hypoxic condition caused by nurse cell formation. Localization of the hypoxic regions by the hypoxia marker, pimonidazole showed T. spiralis infection does not induce a hypoxic condition in nurse cells. These results suggest that the expression of VEGF and thymosin β4 induce angiogensis and the expression of thymosin β4 remained elevated to potentially regulate nurse cell formation and maintenance.

First record of a Kabatana sp. microsporidium infecting fish in the Atlantic Ocean

Two-spotted goby Gobiusculus flavescens from the Swedish Gullmarsfjord regularly present subcutaneous creamy-white patches in the body musculature, associated with Kabatana sp. infection. Analysis of the 16S rRNA gene of the microsporidium showed 98.54% homology with Kabatana newberryi infecting a marine goby from California, indicating that the Swedish microsporidium is either a different strain of K. newberryi or a closely related species. This represents the first record of a Kabatana species in the Atlantic Ocean. The genetic similarity of the 2 microsporidia was paralleled by close infection phenotypes. Infected muscle fibres were swollen compared to adjacent non-infected fibres, and mature spore masses were found throughout the skeletal musculature. No xenoma formation was detected. Since G. flavescens is an established model species in behavioural ecology, the host-parasite system is ideally suited for testing how microsporidian infections affect host behaviour and fitness.

Macrophages and giant cells associated with a microsporidian parasite causing liquefaction of the skeletal muscle of the Norway pout, Trisopterus esmarkii (Nilsson)

. A mature microsporidian infection of the skeletal muscles of Norway pout, Trisopterus esmarkii Nilsson, is described. Infected fish had noticeable liquefaction of the muscle. The white foci of infection contained packed microsporidian spores, and the surrounding infected muscle fibres were degenerate. The host response involved invasion by phagocytic cells and encapsulation by fibroblasts, forming large granulomas. Macrophages, 17–20 μm in diameter, were actively ingesting the spores and typically had a foamy cytoplasm. Some had formed Langhans-type giant cells, 35 μm in diameter, with peripherally arranged nuclei. Sporulation was complete and appeared to have occurred within sporo- phorous vacuoles in which numerous spores were formed. Individual spores measured 2·8 × 1·5 μm and were characterized by irregular electron dense areas, a short polar filament with four coils and a large posterior vacuole with three inclusions.

Cytopathic Effect of Lentogenic Strains of Newcastle Disease Virus in Cultured Chicken Muscle Cells

The ability of lentogenic strains of Newcastle disease virus (NDV) to inhibit the development of embryonic chicken muscle cells in vitro is described. In contrast to the uninfected control, which displayed a thick network of branched multinucleated muscle fibers, cultures infected with NDV exhibited a severely reduced potential to differentiate. The few differentiated muscle fibers that formed in the presence of NDV were poorly developed, displayed an attenuated morphology, and detached from the substrate 2 to 3 days before uninfected cultures showed signs of cell aging. When NDV-infected cultures were stained before muscle fiber detachment with a fluorescent-labeled NDV-specific antibody, the infected muscle fibers reacted strongly relative to neighboring myoblasts and fibroblasts. All six strains of NDV induced a similar inhibitory effect, and no strain-specific differences were detectable. These results indicate that differentiating muscle fibers are highly susceptible to infection by NDV in vitro and that this tissue exhibits an early NDV-induced cytopathic effect.

The pathogenicity and development within the host fish of Myxobolus cyprini Doflein, 1898

Myxobolus cyprini has, until now, been considered an ‘organo-cosmopolitan’ parasite of the common carp (Cyprinus carpio) and less frequently of other carps, producing spores in various organs in small plasmodia and possible in cysts. The present observations of naturally infected common carp fry and two-summer carp have revealed that M. cyprini is a specific muscle parasite, developing intracellularly in the muscle fibres of the skeletal muscle. The sarcoplasm of the infected muscle fibres is filled with developmental stages, followed by spores of M. cyprini, which are held together in a 1–1·5 mm long pseudocyst by the sarcolemma of the muscle fibre. After maturation of the spores and disintegration of the pseudocysts the spores are transported in the bloodstream to different parts of the body where they are retained in the capillaries.

Effect of denervation on coxsackie A virus myositis in mice

Myositis induced by Coxsackie A4 and A9 viruses was investigated in the gastrocnemius muscles of suckling mice and adult mice with denervation. Denervation markedly increased the susceptibility of adult mice to Coxsackie A virus infection, and this effect was initiated as early as 1 day after denervation. Light microscopy demonstrated inflammation and necrosis in the denervated gastrocnemius muscle of adult mice, whereas muscle from the contralateral leg showed only infrequent, mild, focal myositis. Ultrastructually, crystalline arrays of virus particles were seen in the infected muscle fibers and in the phagocytes of both suckling and adult mice. Nuclear alterations, especially in the myotubes, and a characteristic compound membrane-vesicle complex (CMVC) in the sarcoplasm, developed simultaneously. Replicating and fusing myoblasts, activated as part of the regenerative process after denervation, appeared to be closely associated with the susceptibility of muscle to Coxsackie A virus infection.

Development of Brugia pahangi in the flight muscles of Aedes togoi. Ultrastructural changes in the infected muscle fibers and the infecting filarial larvae.

The ultrastructural changes seen in the indirect flight muscles of Aedes togoi infected with Brugia pahangi included the disarray or disruption of the parasitized muscle fiber, the reduction and disorganization of the interfibrillar mitochondria, the reduction of muscle glycogen and, finally, the complete dissolution of the muscle fiber. These changes are restricted to individual parasitized muscle fibers while adjacent non-parasitized fibers retain their usual arrangement. The damage inflicted to these parasitized fibers may be due to the presence and movement of the larvae within the fibrils and the active ingestion of muscle mitochondria by these developing larvae. The damaged muscle may be capable of partial repair as the extent of injury was usually less severe in infected mosquitoes from the 9th day after the infecting feed, when most of the infective larvae had migrated from the thorax to the head and proboscis.

Some Aspects of Cyst Synthesis in Mouse Trichinosis

The total hydroxyproline of the diaphragms of mice infected with T. spiralis increased over that of uninfected about day 25 PI. The rates of incorporation of proline and methionine into protein of infected diaphragms were above those of uninfected after day 13 PI. These findings are discussed in terms of the chronology of trichinella-cyst formation and in terms of changes in the morphology and RNA metabolism of infected muscle fibers. Stewart and Read (1972) demonstrated marked changes in the total ribonucleic acid (RNA) and rates of synthesis of RNA in trichinosed versus uninfected diaphragm muscle. The earliest of these changes occur concomitantly with reported morphological alterations in infected fibers (Ribas-Mujal and Rivera-Pomar, 1968; Fasske and Themann, 1961). Read (1970) reviewed various studies indicating that profound chemical changes occur in host muscle during the early stages of infection with trichinella. Ritterson (1966) and Bruce (1970) have shown that trichinella cysts are composed primarily of collagen. It was the object of the present study to examine certain aspects of the biochemical development of trichinella cysts in host muscle. Changes in total collagen, the rates of incorporation of proline, and the relation of cyst protein synthesis to the total protein synthetic activity of infected versus uninfected diaphragm muscle were investigated. MATERIALS AND METHODS The source of trichinella, method of excystment of the muscle larvae, and the procedure for the infection of experimental animals were those used in previous studies (Stewart and Read, 1972). Male, 5-week-old Swiss white mice (Texas Inbred Mice Co., Houston, Texas) were used in all experiments. To minimize the age disparity between muscle larvae, the anthelminthic methyridine was used to abbreviate the period of infection by adults in the intestines of experimental animals (Stewart and Read, 1972). All experimental animals were infected with 1,000 larvae and methyridine was given on day 11 PI (postinfection) at a dose rate of 500 mg/kilo. Received for publication 6 July 1972. * This work was partially supported by a grant from the NIH (2T01 AI00106). In all experiments the RNA and protein fractions were removed from the muscle by a modified method of Schmidt and Thannhauser (1945). Total RNA was determined by the orcinol method of Dische (1955), protein was assayed by the method of Lowry et al. (1951), and total hydroxyproline was determined by the method of Bergman and Loxley (1963). In the experiments of protein synthesis the diaphragms of infected and uninfected mice were removed, washed briefly in Krebs-Ringers solution containing 25 mh tris-maleate buffer at pH 7.4 (KRT), and placed in 50-ml Erlenmeyer flasks containing 1.25 ,uM 14C-proline (sp. act. = 1.6 ,uCi/tuM) or 2 AuM 35S-methionine (sp. act. = 5 ACi/AuM) and 10 mh glucose in 25 ml KRT. All incubations were for 1 hr at 37 C with oxygen bubbled directly into the medium. After 1 hr diaphragms were removed from the incubation medium, rinsed twice in KRT, blotted on filter paper, and placed in dry test tubes immersed in a dry ice-acetone bath. After all incubations were completed, the diaphragms were homogenized in cold 0.5 N perchloric acid and submitted to the Schmidt-Thannhauser extraction procedure. In experiments on the incorporation of 14Cproline and 35S-methionine, radioactivity of two 0.5-ml aliquots of the protein fraction from each diaphragm was determined in a gas-flow counter. Two-milliliter samples of the protein fraction from each diaphragm collected on selected days were hydrolyzed in 6 N HC1 at 125 C for 1 hr in sealed ampoules. The protein hydrolysates were evaporated to dryness and redissolved in 0.1 N HC1 and the disposition of the radioactivity was determined on a Technicon amino acid analyzer and a Packard Tri-Carb analyzer. It was found that the amount of 14C-proline hydroxylated to hydroxyproline in 1 hr in vitro incubations could not be detected. Therefore, on days 12, 21, and 28 PI 3 infected and 3 uninfected mice were injected intraperitoneally with 4 JtCi of 14C-proline and killed 24 hr after injection of the isotope. The diaphragms of these mice were removed, and an aliquot of the extracted protein fraction was hydrolyzed and the disposition of the label was determined by the above methods. In experiment I determination of the rates of incorporation of proline, total hydroxyproline, and total RNA were carried out on the same experi-

Ribonucleic Acid Metabolism in Mouse Trichinosis

Trichinosed mouse diaphragms showed an increase in total RNA, total DNA, and in the incorporation rates of thymidine over that of control muscle. Larval RNA, protein, /Lg RNA per mg protein, and incorporation rates of thymidine were determined. The incorporation of thymidine by infected diaphragms was adjusted for the larval component. Alterations in DNA metabolism are discussed in terms of the morphological changes and changes in RNA metabolism and rates of incorporation of amino acids by infected muscle. Fasske and Themann (1961), and RibasMujal and Rivera-Pomar (1968) have shown that during the early stages of infection with Trichinella spiralis host muscle fibers undergo numerous morphological changes. Read (1970) has reviewed several studies which demonstrate concomitant alterations in the enzymology of infected fibers. These chemical and morphological changes in infected host muscle are accompanied by marked alterations in the total ribonucleic acid (RNA) and rates of synthesis of RNA (Stewart and Read, 1972a). Immediately following a period of rapid RNA turnover there are increases in the rates of incorporation of amino acids by trichinosed muscle (Stewart and Read, 1972b). An increase in the size of nuclei and nucleoli, as well as a proliferation of mitochondria, in infected muscle fibers has been demonstrated by Fasske and Themann (1961) and Ribas-Mujal and Rivera-Pomar (1968). The present study deals with the chronology and magnitude of change in the total deoxyribonucleic acid (DNA) and rates of synthesis of DNA in infected and uninfected mouse

Notes on Sarcosporidia of Birds in Panama

in the present study. These infected birds were found in a lot of 36 specimens, representing 21 species, examined for Sarcocystis. They were collected in 1952-53 from areas of Panama near the Canal Zone, while the writer was at the Gorgas Hospital Laboratory. Each bird was examined for intestinal and blood parasites. Blocks of tissue routinely were cut from the vital organs and muscle and preserved in 10% formalin for the later preparation of sections which were stained with hematoxylin and eosin. Generally, Sarcocystis infection was noted only during the microscopic examination of striated muscle sections. However, the sarcocysts in the collared aricari were seen while the bird was being skinned. In the one infected aricari of the 4 examined, the back, breast, thigh, and neck nmuscles of the bird contained numerous elongated yellow capsules which were 1 to 1.5 mm long and about half as wide. Some of the capsules were removed, ruptured, and the contents stained with Giemsa's stain after heat fixation. Bananashaped sporozoites, 3 to 4.5 microns long and about 1 micron wide, with subterminal nuclei were demonstrated. In longitudinal sections, the largest sarcocyst was 1.23 mm long and 0.339 mm wide at the point of greatest diameter. The cysts were oval in shape and bounded by a thin membranous wall. Many sarcocysts were measured which ranged from 0.462 to 0.678 mm long and 0.123 to 0.354 mm wide. There was also a large number of very much smaller cysts ranging from 0.046 to 0.154 mm in length and 0.031 to 0.092 mm in width. The large cysts, those over 0.3 mm in diameter, were divided into numerous honeycomb-like chambers at the central portion of the cyst, the Kammerung of Miescher. The central chambers were free of sporozoites and contained only a few scattered ovoid bodies, 2.2 to 4.5 microns in diameter which stained pale pink with eosin. Sporozoites were densely packed into similar chambers in the peripheral portions of the sarcocysts. The Sarcocystis parasitizing the aricari produced marked distortion of the infected muscle fibers. There was no evidence of tissue reaction to the parasites in