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Haycocknema-like nematodes in muscle fibres of a horse
Veterinary Parasitology 139 (2006) 256–261 www.elsevier.com/locate/vetpar
Short communication
Haycocknema-like nematodes in muscle fibres of a horse Johannes Eckert a,*, Pete Ossent b a
b
Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 266a, 8057 Zurich, Switzerland Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 268, 8057 Zurich, Switzerland Received 16 November 2005; received in revised form 20 February 2006; accepted 27 February 2006
Abstract A 14-year-old horse (imported to Switzerland from Ireland 8 years earlier) showed signs of chewing muscle atrophy. A severe chronic myositis, caused by numerous immature and mature female nematodes, was diagnosed in muscle samples obtained by biopsy and subsequently at necropsy. Most of the nematodes had invaded muscle fibres of the masseter, root of the tongue and anterior breast, only a few were found in the intermuscular interstitium. Isolated nematodes and parasite sections were clearly different from muscle larvae of Trichinella spp. but showed morphological similarities to Haycocknema perplexum, a nematode species (order Enoplida, family Robertdollfusidae) recently found in the musculature of a human patient in Australia. However, our material did not allow the precise identification of the nematode genus nor the unequivocal differentiation from Halicephalobus gingivalis. This species infects horses and humans and can cause severe granuloma formation in muscles and many other organ systems, but has never been observed to invade individual muscle fibres. Our findings show that nematodes of another genus than Trichinella may invade muscle fibres of the horse and cause myositis. These nematodes are provisionally regarded as Haycocknema-like. # 2006 Elsevier B.V. All rights reserved. Keywords: Trichinella; Halicephalobus; Haycocknema; Horse; Muscle; Differential diagnosis
1. Introduction To date, the only nematodes known to inhabit the fibres of skeletal muscles in horses are larvae of Trichinella species, including Trichinella spiralis, T. britovi and T. murrelli (Arriaga et al., 1995; Pozio, 2001; Pozio et al., 2002). Adult females and larval stages of the rhabditoid nematode Halicephalobus * Corresponding author. Tel.: +41 44 422 14 54; fax: +41 44 422 14 07. E-mail address: [email protected] (J. Eckert).
gingivalis (order Rhabditida, family Cephalobidae) have been recorded in various equine organs, including musculature (Anderson et al., 1998; Teifke et al., 1998; Weaver et al., 1999; Wollanke et al., 2000, and others). These nematodes were typically found in granulomas in the affected organs, but – to our knowledge – an intra-myofibre location has never been observed. In this communication we describe the invasion of muscle fibres in a horse by nematodes showing characteristics which differ morphologically from both Trichinella and Halicephalobus.
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.02.042
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
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2. Case report A 14-year-old mare imported to Switzerland from Ireland approximately 8 years previously had suffered from increasing inappetence and emaciation for several months and finally showed distinct atrophy of the masticatory muscles leading to partial inability to chew. The histological examination of two biopsies of the masseter muscles revealed signs of myositis and numerous sections of nematodes, most of them within muscle fibres. The findings, however, did not allow the identification of the parasite. The horse was slaughtered because the clinical condition failed to improve and a necropsy was performed. Nematodes were found in squash preparations (pressure glass technique) of muscle samples from the masseter, the root of the tongue and anterior breast. Under low microscopic magnification the parasites were either stretched out lengthways within muscle tissue, were partially curled or showed a bent tail (Figs. 1 and 2). They contained dense, dark granules in the middle section of the body, and there was no capsule around the parasites. Some nematodes could be isolated by peptic digestion of muscle samples from the masseter and the anterior breast but most of them were damaged and the internal structures were only discernible in a small number of specimens. The
Fig. 1. Stretched larval nematode stages in masseter muscle (squash preparation).
Fig. 2. Partially curled larval nematode stages in masseter muscle (squash preparation).
stretched nematodes (n: 9, unfixed in squash preparations or isolated by peptic digestion) had an average body length of 289 mm (253–319 mm) and a maximum width (n: 4) of 15.2 mm (12–19 mm). These stages had a rounded anterior end and a tail ending in a needle-like tip (Fig. 3); in the anterior third of the nematode, indistinct outlines of an oesophagus were visible, but a typical rhabditoid structure could not be discerned. The middle section of the body contained numerous dark granules; indications of sexual differentiation could not be observed. The posterior part of two larger specimens showed a vulva-like slit on a slight elevation and a conical tail ending in a point (Fig. 4a and b). These stages had the following dimensions: body length: of 407–435 mm, oesophagus length 78–82 mm, vulva slit 224–242 mm from the anterior end. Genital organ structures other than the vulva could not be seen. Histological examination of musculature revealed a severe, chronic and active myositis in various parts of the head and breast musculature associated with
Fig. 3. Isolated larval nematode from muscle tissue.
258
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Fig. 4. (a) Anterior end of a female stage from muscle tissue; (b) posterior end of a female stage teased from muscle tissue.
numerous sections of nematodes. Nematodes were neither found in other skeletal muscles (neck, shoulder, sacral region, diaphragm, gluteal and intercostal muscles) nor in various organs (heart, lung, wall of oesophagus, stomach and intestine, liver, kidney, uterus, urinary bladder, or spinal cord). (The brain was not examined.) An inflammatory focus in the musculature is shown in Fig. 5 together with numerous cross sections of nematodes located within myofibres.
Many nematodes appeared to be degenerated and were surrounded by heavy inflammatory reaction. In longitudinal sections slender nematodes (Fig. 6) or thicker forms with internal structures were observed. Figs. 7 and 8 show within a muscle fibre the outline of a larger nematode containing slender parasite forms. Some muscle fibres contained a row of at least five ovoid bodies (approximately 18 mm 7 mm) which match the structure of nematode oocytes or eggs (Fig. 9).
Fig. 5. Histological section of tongue with focal inflammation and numerous cross sections of nematodes within muscle fibres (arrows); hematoxylin–eosin staining.
Fig. 6. Histological section of tongue with a stretched intracellular larval nematode; hematoxylin–eosin staining.
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Fig. 7. Histological section of tongue showing within a muscle fibre a degenerated larger nematode (single arrow) containing slender nematode forms (double arrow); hematoxylin–eosin staining.
259
Fig. 9. Histological section of tongue with a row of oocysts or eggs within the remains of a nematode; hematoxylin–eosin staining.
Semi-thin sections proved the invasion of myofibres by nematodes (Fig. 10). DNA was isolated from four paraffin-embedded muscle tissue sections, containing parts of nematodes, using a commercially available kit according to the manufacturer’s instructions (QIAamp DNA kit, Qiagen, Germany). To check whether DNA amplifiable by PCR was isolated, reactions with primers targeting
conserved regions of the 12 S rRNA gene of animals (Kocher et al., 1989) were performed with 25 ml of the DNA solutions, yielding very faint bands of approximately 400 bp upon gel electrophoresis analysis (not shown). PCR primers that had been used to amplify DNA of Trichinella spp. (Rombout et al., 2001) or H. gingivalis (Nadler et al., 2003) did not yield any amplicons with the DNA solutions from these paraffin sections.
Fig. 8. Histological section of tongue showing within a muscle fibre a degenerated larger nematode (single arrow) containing slender nematode forms (double arrow); periodic acid Schiff reaction (PAS).
Fig. 10. Semi-thin section of masseter muscle with a cross section of a nematode within a muscle fibre; toluidin blue staining.
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J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
3. Discussion The nematodes found in our case are much shorter than muscle larvae of Trichinella spp. and differ clearly in several other features such as body shape and the structure of the oesophagus and intestine. Fully developed muscle larvae of Trichinella (T. spiralis, T. pseudospiralis) are 650–1043 mm long and approximately 28–30 mm in width (Dick, 1983). Their oesophagus is surrounded by characteristic stichocytes, the anterior end is slightly pointed, and the posterior end is blunted. Further nematodes must be considered in differential diagnosis. Third-stage larvae of Ancylostoma caninum are known to invade muscle fibres in dogs (Stoye and Schmelzle, 1986). However, such larvae do not come in question here since they possess typical lateral alae and other features. Recently, a new nematode species, Haycocknema perplexum (order Enoplida, superfamily Muspiceidae, family Robertdollfusidae), was described in Australia as an intra-myofibre parasite in a human patient (Dennett et al., 1998; Spratt et al., 1999). Larval stages (body length 147–152 mm), adult females (body length 277–509 mm) and males (body length 295– 307 mm) were observed within individual muscle fibres, but larval stages were also found in interstital locations (Spratt et al., 1999). In our case larval forms and adult females, but no males, were found; most of them had invaded muscle fibres and a few were located in the intermuscular interstitium. The genus Haycocknema and other species of the Robertdollfusidae exhibit some special features: the oesophagus is atrophied posteriorly and is surrounded by one large dorsal and two subventral oesophageal glands, the intestine is transformed to a ‘‘trophosome’’ filled with dark, refringent granules, and the female has a large ‘‘amorphous cell’’ supporting a granule-filled flask-shaped reservoir in the rectal region. The adult females of H. perplexum can contain eggs or larvae, and are described as viviparous (for more details see Spratt et al., 1999; Spratt and Nicholas, 2002). The nematodes found in our case exhibited features of H. perplexum, including the body dimensions, the indistinct oesophagus, a trophosome-like structrue filled with dark granules, and the location of larval stages and mature females within myofibres. Furthermore, females containing several eggs in the same stage of development
(Fig. 9) or larvae were found in muscle fibres (Fig. 7). However, a definitive diagnosis could not be made because most of the isolated nematodes were degenerated (see above), and the few adult stages available did not allow a detailed identification of internal structures. The genus Haycocknema within the family Robertdollfusidae belongs to the order Enoplida and the suborder Trichinellina. The method of transmission of the Robertdollfusidae is not known; tentative observations suggest that arthropod vectors may be involved (Anderson, 2000). The predominant invasion of the head and breast muscles in our case is rather suggestive for an infection route via the oral mucosa. The genus Halicephalobus includes eight nominal species which were originally isolated from soil, organic material or plants. Only one of these freeliving species, H. gingivalis (syn. Halicephalobus deletrix, Micronema deletrix) has been found to infect occasionally horses (Pohlenz et al., 1981; Liebler et al., 1989; Anderson et al., 1998; Teifke et al., 1998; Weaver et al., 1999; Wollanke et al., 2000; Nadler et al., 2003, and others), zebras (Isaza et al., 2000) and humans (reviewed in Anderson et al., 1998). Analyses of the LSU rDNA sequences have revealed that H. gingivalis is a distinct species consisting of more than one genetically distinct lineage (Nadler et al., 2003). The body dimensions of the larval stages and the adult female nematodes from our case are also similar to H. gingivalis (Pohlenz et al., 1981; Anderson et al., 1998). However, the rhabditoid oesophagus characteristic for H. gingivalis could not be discerned, and neither a ‘‘trophosome’’ filled with dark granules nor the location of larval and mature female stages within myocytes have been observed in this species. Therefore, we regard the nematodes found in our case provisionally as Haycocknema-like. These negative results of our DNA studies may support our diagnosis of a Haycocknema-like nematode but caution is indicated as the only material preserved from this case seems to be ill-suited for PCR analysis due to fixation and embedding for a prolonged period of time.
Acknowledgements The authors thank Dr. Ian Beveridge, Melbourne, and Dr. D.M. Spratt, Canberra, for their advice and
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Dr. A. Mathis, Institute of Parasitology Zurich, for the PCR analysis.
References Anderson, R.C., 2000. Nematode parasites of vertebrates. In: Their Development and Transmission, second ed. CABI Publishing, CAB International, Wallingford, Oxon, 650 pp. Anderson, R.C., Linder, K.E., Peregrine, A.S., 1998. Halicephalobus gingivalis (Stefanski, 1954) from a fatal infection in a horse in Ontario, Canada, with comments on the validity of H. deletrix and a review of the genus. Parasite 5, 255–261. Arriaga, C., Ye´pez-Mulia, L., Viveros, N., Adame, L.A., Zarlenga, D.S., Lichtenfels, R., Benitez, E., Ortega-Pierres, M.G., 1995. Detection of Trichinella spiralis muscle larvae in naturally infected horses. J. Parasitol. 81, 781–783. Dennett, X., Siejka, S., Andrews, J.R.H., Beveridge, I., Spratt, D.M., 1998. Polymyositis caused by a new genus of nematode. Med. J. Aust. 168, 226–227. Dick, T.A., 1983. Species, and intraspecific variation. In: Campbell, W.C. (Ed.), Trichinella and Trichinosis. Plenum Press, New York, pp. 31–73. Isaza, R., Schiller, C.A., Stover, J., Smith, P.J., Greiner, E.C., 2000. Halicephalobus gingivalis (Nematoda) infection in a Grevy’s zebra (Equus grevyi). J. Zoo Wildlife Med. 31, 77–81. Kocher, T.D., Thomans, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Nat. Acad. Sci. U.S.A. 86, 6169–6200. Liebler, E.M., Gerhards, H., Denkhaus, M., Pohlenz, J., 1989. Micronema deletrix als Ursache einer granulomato¨sen Nephritis bei einem Pferd. Dtsch. Tiera¨rztl. Wochenschr. 96, 223–225. Nadler, S.A., Carreno, R.A., Adams, B.J., Kinde, H., Baldwin, J.G., Mundo-Ocampo, M., 2003. Molecular phylogenesis and diagnosis of soil and clinical isolates of Halicephalobus gingivalis
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(Nematoda: Cephalobina: Panagrolaimoidea), an opportunistic pathogen of horses. Int. J. Parasitol. 33, 1115–1125. Pohlenz, J., Eckert, J., Minder, H.P., 1981. Zentralnervo¨se Sto¨rungen beim Pferd, verursacht durch Nematoden der Gattung Micronema (Rhabditida). Berlin Mu¨nch. Tiera¨rztl. Wochenschr. 94, 216–220. Pozio, E., 2001. New patterns of Trichinella infection. Vet. Parasitol. 98, 133–148. Pozio, E., Foggin, C.M., Marucci, G., La Rosa, G., Sacchi, L., Corona, S., Rossi, P., Mukaratirwa, S., 2002. Trichinella zimbabwensis n.sp. (Nematoda), a new non-encapsulated species from crocodiles (Crocodylus niloticus) in Zimbabwe also infecting mammals. Int. J. Parasitol. 32, 1787–1799. Rombout, Y.B., Bosch, S., van der Giessen, J.W., 2001. Detection and identification of eight Trichinella genotypes by reverse line blot hybridization. J. Clin. Microbiol. 39, 642–646. Spratt, D.M., Beveridge, I., Andrews, J.R.H., Dennett, X., 1999. Haycocknema perplexum n.g., n. sp. (Nematoda: Robertdollfusidae): an intramyofibre parasite in man. Syst. Parasitol. 43, 123–131. Spratt, D.M., Nicholas, W.L., 2002. Morphological evidence for the systematic position of the order Muspiceida (Nematoda). Trans. R. Soc. South Aust. 126, 51–62. ¨ ber den Ausbreitungsmodus Stoye, M., Schmelzle, H.-M., 1986. U der Larven von Ancylostoma caninum ERCOLANI 1859 (Ancylostomidae) im definitiven Wirt (Beagle). J. Vet. Med. Ser. B 33, 274–283. Teifke, J.P., Schmidt, E., Traenckner, C.M., Bauer, C., 1998. Halicephalobus (Micronema) deletrix als Ursache einer granulomato¨sen Gingivitis und Osteomyelitis bei einem Pferd. Tiera¨rztl. Prax. 26G, 157–161. Weaver, M.P., Callanan, J.J., Schofield, W.C., Torgerson, P., 1999. Equine verminous granulomatous encephalitis in Ireland due to Micronema deletrix infection. Irish Vet. J. 52, 436–439. Wollanke, B., Gerhards, H., Scha¨ffer, E.H., 2000. Keratouveitis und Makrohaematurie bei einer Infektion mit Micronema deletrix bei einem Pferd. Pferdeheilk 16, 23–29.
Short communication
Haycocknema-like nematodes in muscle fibres of a horse Johannes Eckert a,*, Pete Ossent b a
b
Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 266a, 8057 Zurich, Switzerland Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 268, 8057 Zurich, Switzerland Received 16 November 2005; received in revised form 20 February 2006; accepted 27 February 2006
Abstract A 14-year-old horse (imported to Switzerland from Ireland 8 years earlier) showed signs of chewing muscle atrophy. A severe chronic myositis, caused by numerous immature and mature female nematodes, was diagnosed in muscle samples obtained by biopsy and subsequently at necropsy. Most of the nematodes had invaded muscle fibres of the masseter, root of the tongue and anterior breast, only a few were found in the intermuscular interstitium. Isolated nematodes and parasite sections were clearly different from muscle larvae of Trichinella spp. but showed morphological similarities to Haycocknema perplexum, a nematode species (order Enoplida, family Robertdollfusidae) recently found in the musculature of a human patient in Australia. However, our material did not allow the precise identification of the nematode genus nor the unequivocal differentiation from Halicephalobus gingivalis. This species infects horses and humans and can cause severe granuloma formation in muscles and many other organ systems, but has never been observed to invade individual muscle fibres. Our findings show that nematodes of another genus than Trichinella may invade muscle fibres of the horse and cause myositis. These nematodes are provisionally regarded as Haycocknema-like. # 2006 Elsevier B.V. All rights reserved. Keywords: Trichinella; Halicephalobus; Haycocknema; Horse; Muscle; Differential diagnosis
1. Introduction To date, the only nematodes known to inhabit the fibres of skeletal muscles in horses are larvae of Trichinella species, including Trichinella spiralis, T. britovi and T. murrelli (Arriaga et al., 1995; Pozio, 2001; Pozio et al., 2002). Adult females and larval stages of the rhabditoid nematode Halicephalobus * Corresponding author. Tel.: +41 44 422 14 54; fax: +41 44 422 14 07. E-mail address: [email protected] (J. Eckert).
gingivalis (order Rhabditida, family Cephalobidae) have been recorded in various equine organs, including musculature (Anderson et al., 1998; Teifke et al., 1998; Weaver et al., 1999; Wollanke et al., 2000, and others). These nematodes were typically found in granulomas in the affected organs, but – to our knowledge – an intra-myofibre location has never been observed. In this communication we describe the invasion of muscle fibres in a horse by nematodes showing characteristics which differ morphologically from both Trichinella and Halicephalobus.
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.02.042
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
257
2. Case report A 14-year-old mare imported to Switzerland from Ireland approximately 8 years previously had suffered from increasing inappetence and emaciation for several months and finally showed distinct atrophy of the masticatory muscles leading to partial inability to chew. The histological examination of two biopsies of the masseter muscles revealed signs of myositis and numerous sections of nematodes, most of them within muscle fibres. The findings, however, did not allow the identification of the parasite. The horse was slaughtered because the clinical condition failed to improve and a necropsy was performed. Nematodes were found in squash preparations (pressure glass technique) of muscle samples from the masseter, the root of the tongue and anterior breast. Under low microscopic magnification the parasites were either stretched out lengthways within muscle tissue, were partially curled or showed a bent tail (Figs. 1 and 2). They contained dense, dark granules in the middle section of the body, and there was no capsule around the parasites. Some nematodes could be isolated by peptic digestion of muscle samples from the masseter and the anterior breast but most of them were damaged and the internal structures were only discernible in a small number of specimens. The
Fig. 1. Stretched larval nematode stages in masseter muscle (squash preparation).
Fig. 2. Partially curled larval nematode stages in masseter muscle (squash preparation).
stretched nematodes (n: 9, unfixed in squash preparations or isolated by peptic digestion) had an average body length of 289 mm (253–319 mm) and a maximum width (n: 4) of 15.2 mm (12–19 mm). These stages had a rounded anterior end and a tail ending in a needle-like tip (Fig. 3); in the anterior third of the nematode, indistinct outlines of an oesophagus were visible, but a typical rhabditoid structure could not be discerned. The middle section of the body contained numerous dark granules; indications of sexual differentiation could not be observed. The posterior part of two larger specimens showed a vulva-like slit on a slight elevation and a conical tail ending in a point (Fig. 4a and b). These stages had the following dimensions: body length: of 407–435 mm, oesophagus length 78–82 mm, vulva slit 224–242 mm from the anterior end. Genital organ structures other than the vulva could not be seen. Histological examination of musculature revealed a severe, chronic and active myositis in various parts of the head and breast musculature associated with
Fig. 3. Isolated larval nematode from muscle tissue.
258
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Fig. 4. (a) Anterior end of a female stage from muscle tissue; (b) posterior end of a female stage teased from muscle tissue.
numerous sections of nematodes. Nematodes were neither found in other skeletal muscles (neck, shoulder, sacral region, diaphragm, gluteal and intercostal muscles) nor in various organs (heart, lung, wall of oesophagus, stomach and intestine, liver, kidney, uterus, urinary bladder, or spinal cord). (The brain was not examined.) An inflammatory focus in the musculature is shown in Fig. 5 together with numerous cross sections of nematodes located within myofibres.
Many nematodes appeared to be degenerated and were surrounded by heavy inflammatory reaction. In longitudinal sections slender nematodes (Fig. 6) or thicker forms with internal structures were observed. Figs. 7 and 8 show within a muscle fibre the outline of a larger nematode containing slender parasite forms. Some muscle fibres contained a row of at least five ovoid bodies (approximately 18 mm 7 mm) which match the structure of nematode oocytes or eggs (Fig. 9).
Fig. 5. Histological section of tongue with focal inflammation and numerous cross sections of nematodes within muscle fibres (arrows); hematoxylin–eosin staining.
Fig. 6. Histological section of tongue with a stretched intracellular larval nematode; hematoxylin–eosin staining.
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Fig. 7. Histological section of tongue showing within a muscle fibre a degenerated larger nematode (single arrow) containing slender nematode forms (double arrow); hematoxylin–eosin staining.
259
Fig. 9. Histological section of tongue with a row of oocysts or eggs within the remains of a nematode; hematoxylin–eosin staining.
Semi-thin sections proved the invasion of myofibres by nematodes (Fig. 10). DNA was isolated from four paraffin-embedded muscle tissue sections, containing parts of nematodes, using a commercially available kit according to the manufacturer’s instructions (QIAamp DNA kit, Qiagen, Germany). To check whether DNA amplifiable by PCR was isolated, reactions with primers targeting
conserved regions of the 12 S rRNA gene of animals (Kocher et al., 1989) were performed with 25 ml of the DNA solutions, yielding very faint bands of approximately 400 bp upon gel electrophoresis analysis (not shown). PCR primers that had been used to amplify DNA of Trichinella spp. (Rombout et al., 2001) or H. gingivalis (Nadler et al., 2003) did not yield any amplicons with the DNA solutions from these paraffin sections.
Fig. 8. Histological section of tongue showing within a muscle fibre a degenerated larger nematode (single arrow) containing slender nematode forms (double arrow); periodic acid Schiff reaction (PAS).
Fig. 10. Semi-thin section of masseter muscle with a cross section of a nematode within a muscle fibre; toluidin blue staining.
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J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
3. Discussion The nematodes found in our case are much shorter than muscle larvae of Trichinella spp. and differ clearly in several other features such as body shape and the structure of the oesophagus and intestine. Fully developed muscle larvae of Trichinella (T. spiralis, T. pseudospiralis) are 650–1043 mm long and approximately 28–30 mm in width (Dick, 1983). Their oesophagus is surrounded by characteristic stichocytes, the anterior end is slightly pointed, and the posterior end is blunted. Further nematodes must be considered in differential diagnosis. Third-stage larvae of Ancylostoma caninum are known to invade muscle fibres in dogs (Stoye and Schmelzle, 1986). However, such larvae do not come in question here since they possess typical lateral alae and other features. Recently, a new nematode species, Haycocknema perplexum (order Enoplida, superfamily Muspiceidae, family Robertdollfusidae), was described in Australia as an intra-myofibre parasite in a human patient (Dennett et al., 1998; Spratt et al., 1999). Larval stages (body length 147–152 mm), adult females (body length 277–509 mm) and males (body length 295– 307 mm) were observed within individual muscle fibres, but larval stages were also found in interstital locations (Spratt et al., 1999). In our case larval forms and adult females, but no males, were found; most of them had invaded muscle fibres and a few were located in the intermuscular interstitium. The genus Haycocknema and other species of the Robertdollfusidae exhibit some special features: the oesophagus is atrophied posteriorly and is surrounded by one large dorsal and two subventral oesophageal glands, the intestine is transformed to a ‘‘trophosome’’ filled with dark, refringent granules, and the female has a large ‘‘amorphous cell’’ supporting a granule-filled flask-shaped reservoir in the rectal region. The adult females of H. perplexum can contain eggs or larvae, and are described as viviparous (for more details see Spratt et al., 1999; Spratt and Nicholas, 2002). The nematodes found in our case exhibited features of H. perplexum, including the body dimensions, the indistinct oesophagus, a trophosome-like structrue filled with dark granules, and the location of larval stages and mature females within myofibres. Furthermore, females containing several eggs in the same stage of development
(Fig. 9) or larvae were found in muscle fibres (Fig. 7). However, a definitive diagnosis could not be made because most of the isolated nematodes were degenerated (see above), and the few adult stages available did not allow a detailed identification of internal structures. The genus Haycocknema within the family Robertdollfusidae belongs to the order Enoplida and the suborder Trichinellina. The method of transmission of the Robertdollfusidae is not known; tentative observations suggest that arthropod vectors may be involved (Anderson, 2000). The predominant invasion of the head and breast muscles in our case is rather suggestive for an infection route via the oral mucosa. The genus Halicephalobus includes eight nominal species which were originally isolated from soil, organic material or plants. Only one of these freeliving species, H. gingivalis (syn. Halicephalobus deletrix, Micronema deletrix) has been found to infect occasionally horses (Pohlenz et al., 1981; Liebler et al., 1989; Anderson et al., 1998; Teifke et al., 1998; Weaver et al., 1999; Wollanke et al., 2000; Nadler et al., 2003, and others), zebras (Isaza et al., 2000) and humans (reviewed in Anderson et al., 1998). Analyses of the LSU rDNA sequences have revealed that H. gingivalis is a distinct species consisting of more than one genetically distinct lineage (Nadler et al., 2003). The body dimensions of the larval stages and the adult female nematodes from our case are also similar to H. gingivalis (Pohlenz et al., 1981; Anderson et al., 1998). However, the rhabditoid oesophagus characteristic for H. gingivalis could not be discerned, and neither a ‘‘trophosome’’ filled with dark granules nor the location of larval and mature female stages within myocytes have been observed in this species. Therefore, we regard the nematodes found in our case provisionally as Haycocknema-like. These negative results of our DNA studies may support our diagnosis of a Haycocknema-like nematode but caution is indicated as the only material preserved from this case seems to be ill-suited for PCR analysis due to fixation and embedding for a prolonged period of time.
Acknowledgements The authors thank Dr. Ian Beveridge, Melbourne, and Dr. D.M. Spratt, Canberra, for their advice and
J. Eckert, P. Ossent / Veterinary Parasitology 139 (2006) 256–261
Dr. A. Mathis, Institute of Parasitology Zurich, for the PCR analysis.
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