Hostname: page-component-586b7cd67f-g8jcs Total loading time: 0 Render date: 2024-11-28T03:35:38.321Z Has data issue: false hasContentIssue false
  • English
  • Français

Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces

Published online by Cambridge University Press:  18 November 2004

D. TRAVERSA ,
A. GIANGASPERO ,
R. IORIO ,
D. OTRANTO ,
B. PAOLETTI  and
R. B. GASSER
D. TRAVERSA
Affiliation:
Department of Biomedical Comparative Sciences, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
A. GIANGASPERO
Affiliation:
Department of Production Science, Engineering, Mechanics and Economy, Faculty of Agronomy, University of Foggia, Foggia, Italy
R. IORIO
Affiliation:
Department of Biomedical Comparative Sciences, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
D. OTRANTO
Affiliation:
Department of Animal Health and Welfare, Faculty of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy
B. PAOLETTI
Affiliation:
Department of Biomedical Comparative Sciences, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
R. B. GASSER
Affiliation:
Department of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia
Get access Rights & Permissions [Opens in a new window]

Abstract

Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n=46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n=19) or H. muscae (n=4), and others (n=7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3·3%) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100% and a sensitivity of ~97% (being able to specifically detect as little as ~0·02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.

Keywords

coprological diagnosisepidemiologyhorseHabronema microstomaHabronema muscaesecond internal transcribed spacer of ribosomal DNAsemi-nested PCR
Type
Research Article
Information
Parasitology , Volume 129 , Issue 6 , December 2004 , pp. 733 - 739
Copyright
© 2004 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

ANDERSON, R. C. ( 2000). The superfamily Habronematoidea. In Nematode Parasites of Vertebrates. Their Development and Transmission, 2nd Edn, pp. 428–430. CABI Publishing, Guilford, UK.
BORGSTEEDE, F. H. & VAN BEEK, G. ( 1998). Parasites of stomach and small intestine of 70 horses slaughtered in The Netherlands. Veterinary Quarterly 20, 31–34.CrossRefGoogle Scholar
BUCKNELL, D. G., GASSER, R. B. & BEVERIDGE, I. ( 1995). The prevalence and epidemiology of gastrointestinal parasites of horses in Victoria, Australia. International Journal for Parasitology 25, 711–724.CrossRefGoogle Scholar
CHAFFIN, M. K., SCHUMACHER, J. & McMULLAN, W. C. ( 1995). Cutaneous pythiosis in the horse. Veterinary Clinics of North America: Equine Practice 11, 91–103.CrossRefGoogle Scholar
DE JESUS, Z. ( 1963). Observations of habronemiasis in horses. Philippines Veterinary Medicine 2, 133–152.Google Scholar
DUNN, A. M. ( 1969). Habronemiasis and Drascheiasis (sic. Draschiasis). In Veterinary Helminthology, pp. 199–200. W. Heinemann Medical Books Ltd, London, UK.
EUZEBY, J. ( 1961). Les Maladies Vermineuses des Animaux Domestiques et leurs Incidences sur la Pathologie Humaine. Maladies dues aux Nemathelminthes. Fascicule Premier. Vigot Frères, Paris, France.
EUZEBY, J. ( 1981). Diagnostic expérimental des helminthoses animales. Travaux pratiques d'helminthologie vétérinaire. Livre 1. Edition “Informations Techniques des Services Vétérinaires”, Paris, France.
GASSER, R. B. ( 1999). PCR-based technology in veterinary parasitology. Veterinary Parasitology 84, 229–258.CrossRefGoogle Scholar
GASSER, R. B., ZHU, X. Q., BEVERIDGE, I. & CHILTON, N. B. ( 2001). Mutation scanning analysis of sequence heterogeneity in the second internal transcribed spacer (rDNA) within some members of the Hypodontus macropi (Nematoda: Strongyloidea) complex. Electrophoresis 22, 1076–1085.3.0.CO;2-8>CrossRefGoogle Scholar
HASS, K. ( 1979). Equine parasitism. Veterinary Medicine and Small Animal Clinician 74, 980–988.Google Scholar
HUNG, G.-C., GASSER, R. B., BEVERIDGE, I. & CHILTON, N. B. ( 1999). Species-specific amplification by PCR of ribosomal DNA from some equine strongyles. Parasitology 119, 69–80.CrossRefGoogle Scholar
INZANA, T. J. & CARTER, G. R. ( 1990). Fungi causing systemic or deep infections. In Diagnostic Procedures in Veterinary Bacteriology and Mycology, 5th Edn (ed. Carter, G. R. & Cole, J. R.), pp. 433–455. Academic Press Inc., San Diego, CA, USA.CrossRef
LICHTENFELS, J. R. ( 1975). Helminths of domestic equids. Proceedings of the Helminthological Society of Washington 42, 1–92.Google Scholar
LYONS, E. T., TOLLIVER, S. C., DRUDGE, J. H., SWERCZEK, T. W. & CROWE, M. W. ( 1983). Parasites in Kentucky thoroughbreds at necropsy: emphasis on stomach worms and tapeworms. American Journal of Veterinary Research 44, 839–844.Google Scholar
LYONS, E. T., TOLLIVER, S. C., DRUDGE, J. H., SWERCZEK, T. W. & CROWE, M. W. ( 1987). Common internal parasites found in the stomach, large intestine and cranial mesenteric artery of thoroughbreds in Kentucky at necropsy (1985 to 1986). American Journal of Veterinary Research 48, 268–273.Google Scholar
LYONS, E. T., TOLLIVER, S. C., STAMPER, S., DRUDGE, J. H., GRANSTROM, D. E. & COLLINS, S. S. ( 1994). Transmission of some species of internal parasites in horses born in 1990, 1991 and 1992 in the same pasture on a farm in central Kentucky. Veterinary Parasitology 52, 257–269.CrossRefGoogle Scholar
OWEN, J. & SLOCOMBE, D. ( 1985). Pathogenesis of helminths in equines. Veterinary Parasitology 18, 139–153.CrossRefGoogle Scholar
PANDEY, V. S., OUHELLI, H. & VERHULST, A. ( 1992). Epidemiological observations on stomach worms of donkeys in Morocco. Veterinary Research Communications 16, 273–279.CrossRefGoogle Scholar
PASCOE, R. R. ( 1990). A Colour Atlas of Equine Dermatology. Wolfe Publishing Ltd, Ipswich, UK.
RICCI, M. & SABATINI, A. ( 1992). Notizie sugli elminti parassiti del cieco e del colon degli equidi in Italia. Parassitologia 34, 53–60.Google Scholar
ROUBAUD, E. & DESCAUZEAUX, J. ( 1922). Evolution de l'Habronema muscae Carter chez la mouche domestique et de l'H. microstomum Schneider chez le stomoxe (Note préliminaire) comme Agent Vecteur des Habronèmosis d'Équides. Bulletin de la Société de Pathologie Exotique 15, 572–574.Google Scholar
SCHMIDT, G. D. ( 1986). Handbook of Tapeworm Identification. CRC Press, Boca Raton, Florida, USA.
SOULSBY, E. J. L. ( 1982). Helminths, Arthropods and Protozoa of Domesticated Animals, 7th Edn. Baillière, Tindall and Cassell, Eastbourne, UK.
THOMPSON, J. D., GIBSON, T. J., PLEWNIAK, F., JEANMOUGIN, F. & HIGGINS, D. G. ( 1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882.CrossRefGoogle Scholar
TOLLIVER, S. G. ( 2000). A Practical Method of Identification of the North American Cyathostomes (Small Strongyles) in Equids in Kentucky. Published by the Kentucky Agricultural Experiment Station-College of Agriculture, University of Kentucky, Lexington, Kentucky, USA.
TRAVERSA, D., GIANGASPERO, A., GALLI, P., PAOLETTI, B., OTRANTO, D. & GASSER, R. B. ( 2004). Specific identification of Habronema microstoma and Habronema muscae (Spirurida, Habronematidae) by PCR using markers in ribosomal DNA. Molecular and Cellular Probes 18, 215–221.CrossRefGoogle Scholar
VERWEIJ, J. J., PIT, D. S., VAN LIESHOUT, L., BAETA, S. M., DERY, G. D., GASSER, R. B. & POLDERMAN, A. M. ( 2001). Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Tropical Medicine and International Health 6, 726–731.CrossRefGoogle Scholar
VERWEIJ, J. J., POLDERMAN, A. M., WIMMENHOVE, M. C. & GASSER, R. B. ( 2000). PCR assay for the specific amplification of Oesophagostomum bifurcum DNA from human faeces. International Journal for Parasitology 30, 137–142.CrossRefGoogle Scholar
ZUMPT, F. ( 1965). Myiasis in Man and Animals in the Old World. Butterworths, London, UK.