Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Jul;35(7):1740–1745. doi: 10.1128/jcm.35.7.1740-1745.1997

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

M S Ho 1, B C Barr 1, A F Tarantal 1, L T Lai 1, A G Hendrickx 1, A E Marsh 1, K W Sverlow 1, A E Packham 1, P A Conrad 1
PMCID: PMC229832  PMID: 9196184

Abstract

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.

Full Text

The Full Text of this article is available as a PDF (202.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. L., Blanchard P. C., Barr B. C., Dubey J. P., Hoffman R. L., Conrad P. A. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J Am Vet Med Assoc. 1991 Jan 15;198(2):241–244. [PubMed] [Google Scholar]
  2. Anderson M. L., Palmer C. W., Thurmond M. C., Picanso J. P., Blanchard P. C., Breitmeyer R. E., Layton A. W., McAllister M., Daft B., Kinde H. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J Am Vet Med Assoc. 1995 Nov 1;207(9):1206–1210. [PubMed] [Google Scholar]
  3. Anderson M. L., Reynolds J. P., Rowe J. D., Sverlow K. W., Packham A. E., Barr B. C., Conrad P. A. Evidence of vertical transmission of Neospora sp infection in dairy cattle. J Am Vet Med Assoc. 1997 Apr 15;210(8):1169–1172. [PubMed] [Google Scholar]
  4. Barr B. C., Anderson M. L., Blanchard P. C., Daft B. M., Kinde H., Conrad P. A. Bovine fetal encephalitis and myocarditis associated with protozoa. Vet Rec. 1990 Feb 3;126(5):120–121. [PubMed] [Google Scholar]
  5. Barr B. C., Anderson M. L., Dubey J. P., Conrad P. A. Neospora-like protozoal infections associated with bovine abortions. Vet Pathol. 1991 Mar;28(2):110–116. doi: 10.1177/030098589102800202. [DOI] [PubMed] [Google Scholar]
  6. Barr B. C., Anderson M. L., Woods L. W., Dubey J. P., Conrad P. A. Neospora-like protozoal infections associated with abortion in goats. J Vet Diagn Invest. 1992 Jul;4(3):365–367. doi: 10.1177/104063879200400331. [DOI] [PubMed] [Google Scholar]
  7. Barr B. C., Conrad P. A., Breitmeyer R., Sverlow K., Anderson M. L., Reynolds J., Chauvet A. E., Dubey J. P., Ardans A. A. Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: four cases (1990-1992). J Am Vet Med Assoc. 1993 Jan 1;202(1):113–117. [PubMed] [Google Scholar]
  8. Barr B. C., Conrad P. A., Dubey J. P., Anderson M. L. Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity. J Vet Diagn Invest. 1991 Jan;3(1):39–46. doi: 10.1177/104063879100300109. [DOI] [PubMed] [Google Scholar]
  9. Barr B. C., Conrad P. A., Sverlow K. W., Tarantal A. F., Hendrickx A. G. Experimental fetal and transplacental Neospora infection in the nonhuman primate. Lab Invest. 1994 Aug;71(2):236–242. [PubMed] [Google Scholar]
  10. Barr B. C., Rowe J. D., Sverlow K. W., BonDurant R. H., Ardans A. A., Oliver M. N., Conrad P. A. Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate. J Vet Diagn Invest. 1994 Apr;6(2):207–215. doi: 10.1177/104063879400600212. [DOI] [PubMed] [Google Scholar]
  11. Bjerkås I., Mohn S. F., Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd. 1984;70(2):271–274. doi: 10.1007/BF00942230. [DOI] [PubMed] [Google Scholar]
  12. Bretagne S., Costa J. M., Vidaud M., Tran J., Nhieu V., Fleury-Feith J. Detection of Toxoplasma gondii by competitive DNA amplification of bronchoalveolar lavage samples. J Infect Dis. 1993 Dec;168(6):1585–1588. doi: 10.1093/infdis/168.6.1585. [DOI] [PubMed] [Google Scholar]
  13. Buxton D., Finlayson J. Experimental infection of pregnant sheep with Toxoplasma gondii: pathological and immunological observations on the placenta and foetus. J Comp Pathol. 1986 May;96(3):319–333. doi: 10.1016/0021-9975(86)90052-6. [DOI] [PubMed] [Google Scholar]
  14. Cole R. A., Lindsay D. S., Blagburn B. L., Sorjonen D. C., Dubey J. P. Vertical transmission of Neospora caninum in dogs. J Parasitol. 1995 Apr;81(2):208–211. [PubMed] [Google Scholar]
  15. Conrad P. A., Barr B. C., Sverlow K. W., Anderson M., Daft B., Kinde H., Dubey J. P., Munson L., Ardans A. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology. 1993 Apr;106(Pt 3):239–249. doi: 10.1017/s0031182000075065. [DOI] [PubMed] [Google Scholar]
  16. Cuddon P., Lin D. S., Bowman D. D., Lindsay D. S., Miller T. K., Duncan I. D., deLahunta A., Cummings J., Suter M., Cooper B. Neospora caninum infection in English Springer Spaniel littermates. Diagnostic evaluation and organism isolation. J Vet Intern Med. 1992 Nov-Dec;6(6):325–332. doi: 10.1111/j.1939-1676.1992.tb00364.x. [DOI] [PubMed] [Google Scholar]
  17. Dubey J. P., Acland H. M., Hamir A. N. Neospora caninum (Apicomplexa) in a stillborn goat. J Parasitol. 1992 Jun;78(3):532–534. [PubMed] [Google Scholar]
  18. Dubey J. P., Carpenter J. L., Speer C. A., Topper M. J., Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc. 1988 May 1;192(9):1269–1285. [PubMed] [Google Scholar]
  19. Dubey J. P., Hartley W. J., Lindsay D. S., Topper M. J. Fatal congenital Neospora caninum infection in a lamb. J Parasitol. 1990 Feb;76(1):127–130. [PubMed] [Google Scholar]
  20. Dubey J. P., Hattel A. L., Lindsay D. S., Topper M. J. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J Am Vet Med Assoc. 1988 Nov 15;193(10):1259–1263. [PubMed] [Google Scholar]
  21. Dubey J. P., Koestner A., Piper R. C. Repeated transplacental transmission of Neospora caninum in dogs. J Am Vet Med Assoc. 1990 Oct 1;197(7):857–860. [PubMed] [Google Scholar]
  22. Dubey J. P., Lindsay D. S., Anderson M. L., Davis S. W., Shen S. K. Induced transplacental transmission of Neospora caninum in cattle. J Am Vet Med Assoc. 1992 Sep 1;201(5):709–713. [PubMed] [Google Scholar]
  23. Dubey J. P., Lindsay D. S. Neospora caninum induced abortion in sheep. J Vet Diagn Invest. 1990 Jul;2(3):230–233. doi: 10.1177/104063879000200316. [DOI] [PubMed] [Google Scholar]
  24. Dubey J. P., Lindsay D. S. Transplacental Neospora caninum infection in dogs. Am J Vet Res. 1989 Sep;50(9):1578–1579. [PubMed] [Google Scholar]
  25. Dubey J. P., Porterfield M. L. Neospora caninum (Apicomplexa) in an aborted equine fetus. J Parasitol. 1990 Oct;76(5):732–734. [PubMed] [Google Scholar]
  26. Ellis J., Luton K., Baverstock P. R., Brindley P. J., Nimmo K. A., Johnson A. M. The phylogeny of Neospora caninum. Mol Biochem Parasitol. 1994 Apr;64(2):303–311. doi: 10.1016/0166-6851(94)00033-6. [DOI] [PubMed] [Google Scholar]
  27. Frenkel J. K. Toxoplasmosis in human beings. J Am Vet Med Assoc. 1990 Jan 15;196(2):240–248. [PubMed] [Google Scholar]
  28. Greig B., Rossow K. D., Collins J. E., Dubey J. P. Neospora caninum pneumonia in an adult dog. J Am Vet Med Assoc. 1995 Apr 1;206(7):1000–1001. [PubMed] [Google Scholar]
  29. Guo Z. G., Johnson A. M. Genetic comparison of Neospora caninum with Toxoplasma and Sarcocystis by random amplified polymorphic DNA-polymerase chain reaction. Parasitol Res. 1995;81(5):365–370. doi: 10.1007/BF00931495. [DOI] [PubMed] [Google Scholar]
  30. Guy E. C., Joynson D. H. Potential of the polymerase chain reaction in the diagnosis of active Toxoplasma infection by detection of parasite in blood. J Infect Dis. 1995 Jul;172(1):319–322. doi: 10.1093/infdis/172.1.319. [DOI] [PubMed] [Google Scholar]
  31. Ho M. S., Barr B. C., Marsh A. E., Anderson M. L., Rowe J. D., Tarantal A. F., Hendrickx A. G., Sverlow K., Dubey J. P., Conrad P. A. Identification of bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridization. J Clin Microbiol. 1996 May;34(5):1203–1208. doi: 10.1128/jcm.34.5.1203-1208.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Holliman R. E., Johnson J. D., Gillespie S. H., Johnson M. A., Squire S. B., Savva D. New methods in the diagnosis and management of cerebral toxoplasmosis associated with the acquired immune deficiency syndrome. J Infect. 1991 May;22(3):281–285. doi: 10.1016/s0163-4453(05)80013-1. [DOI] [PubMed] [Google Scholar]
  33. Holliman R. E., Johnson J. D., Savva D. Diagnosis of cerebral toxoplasmosis in association with AIDS using the polymerase chain reaction. Scand J Infect Dis. 1990;22(2):243–244. doi: 10.3109/00365549009037911. [DOI] [PubMed] [Google Scholar]
  34. Holmdahl O. J., Mattsson J. G. Rapid and sensitive identification of Neospora caninum by in vitro amplification of the internal transcribed spacer 1. Parasitology. 1996 Feb;112(Pt 2):177–182. doi: 10.1017/s0031182000084742. [DOI] [PubMed] [Google Scholar]
  35. Holmdahl O. J., Mattsson J. G., Uggla A., Johansson K. E. The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences. FEMS Microbiol Lett. 1994 Jun 1;119(1-2):187–192. doi: 10.1111/j.1574-6968.1994.tb06887.x. [DOI] [PubMed] [Google Scholar]
  36. Jacobson L. S., Jardine J. E. Neospora caninum infection in three Labrador littermates. J S Afr Vet Assoc. 1993 Mar;64(1):47–51. [PubMed] [Google Scholar]
  37. Jardine J. E., Dubey J. P. Canine neosporosis in South Africa. Vet Parasitol. 1992 Oct;44(3-4):291–294. doi: 10.1016/0304-4017(92)90124-r. [DOI] [PubMed] [Google Scholar]
  38. Johnson J. D., Butcher P. D., Savva D., Holliman R. E. Application of the polymerase chain reaction to the diagnosis of human toxoplasmosis. J Infect. 1993 Mar;26(2):147–158. doi: 10.1016/0163-4453(93)92788-x. [DOI] [PubMed] [Google Scholar]
  39. Lally N. C., Jenkins M. C., Dubey J. P. Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene. Mol Biochem Parasitol. 1996 Jan;75(2):169–178. doi: 10.1016/0166-6851(95)02530-8. [DOI] [PubMed] [Google Scholar]
  40. Lindsay D. S., Dubey J. P. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am J Vet Res. 1989 Nov;50(11):1981–1983. [PubMed] [Google Scholar]
  41. Marsh A. E., Barr B. C., Sverlow K., Ho M., Dubey J. P., Conrad P. A. Sequence analysis and comparison of ribosomal DNA from bovine Neospora to similar coccidial parasites. J Parasitol. 1995 Aug;81(4):530–535. [PubMed] [Google Scholar]
  42. Schoondermark-Van de Ven E., Melchers W., Galama J., Camps W., Eskes T., Meuwissen J. Congenital toxoplasmosis: an experimental study in rhesus monkeys for transmission and prenatal diagnosis. Exp Parasitol. 1993 Sep;77(2):200–211. doi: 10.1006/expr.1993.1077. [DOI] [PubMed] [Google Scholar]
  43. Shibata H., Rai S. K., Satoh M., Murakoso K., Sumi K., Uga S., Matsumura T., Matsuoka A. The use of PCR in detecting toxoplasma parasites in the blood and brains of mice experimentally infected with Toxoplasma gondii. Kansenshogaku Zasshi. 1995 Feb;69(2):158–163. doi: 10.11150/kansenshogakuzasshi1970.69.158. [DOI] [PubMed] [Google Scholar]
  44. Thilsted J. P., Dubey J. P. Neosporosis-like abortions in a herd of dairy cattle. J Vet Diagn Invest. 1989 Jul;1(3):205–209. doi: 10.1177/104063878900100301. [DOI] [PubMed] [Google Scholar]
  45. Uggla A., Dubey J. P., Lundmark G., Olson P. Encephalomyelitis and myositis in a boxer puppy due to a Neospora-like infection. Vet Parasitol. 1989 Jul 15;32(2-3):255–260. doi: 10.1016/0304-4017(89)90125-8. [DOI] [PubMed] [Google Scholar]
  46. Wong M. M., Kozek W. J., Karr S. L., Jr, Brayton M. A., Theis J. H., Hendrickx A. G. Experimental congenital infection of Toxoplasma gondii in Macaca arctoides. Asian J Infect Dis. 1979 Jun;3(2):61–67. [PubMed] [Google Scholar]
  47. Wouda W., van den Ingh T. S., van Knapen F., Sluyter F. J., Koeman J. P., Dubey J. P. Neospora abortus bij het rund in Nederland. Tijdschr Diergeneeskd. 1992 Oct 15;117(20):599–602. [PubMed] [Google Scholar]
  48. Yamage M., Flechtner O., Gottstein B. Neospora caninum: specific oligonucleotide primers for the detection of brain "cyst" DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J Parasitol. 1996 Apr;82(2):272–279. [PubMed] [Google Scholar]
  49. van de Ven E., Melchers W., Galama J., Camps W., Meuwissen J. Identification of Toxoplasma gondii infections by BI gene amplification. J Clin Microbiol. 1991 Oct;29(10):2120–2124. doi: 10.1128/jcm.29.10.2120-2124.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES