title: Coccidian oöcysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA.
abstract: Preservation of the exogenous oöcyst stage of coccidian parasites (phylum Apicomplexa N.D. Levine, 1970) as type-specimens of newly described species has long been problematical. Conventional fixatives have proved unsatisfactory, and compromises such as embedding oöcysts in resin or photographing them are not entirely appropriate for various reasons. As an alternative, chilled potassium dichromate solution (normally used in the laboratory to prevent putrefaction of temporary preparations of live oöcysts) has been tested as a long-term preservative of sporulated oöcysts of Eimeria brunetti P.P. Levine, 1942, E. maxima Tyzzer, 1929, E. mitis Tyzzer, 1929, E. necatrix Johnson, 1930, E. praecox Johnson, 1930 and E. tenella (Railliet & Lucet, 1891) (suborder Eimeriorina Léger, 1911; family Eimeriidae Minchin, 1903). Oöcysts from faeces of chickens Gallus gallus (Linnaeus) were placed in 2.5% w/v aqueous potassium dichromate solution (PDS) and stored in the dark at 4 +/- 2 degrees C. After 23 years in storage, oöcysts of each species were administered orally to chickens and failed to initiate infections, indicating that the oöcysts were dead. Nevertheless, after about 24 years, DNA was still recoverable from the oöcysts, and the original species identifications made by classic parasitological methods were confirmed by polymerase chain reaction assays. Furthermore, after almost 25 years, microscopical examination revealed that the walls and internal structures remained well preserved in 83-98% of the oöcysts of the six species investigated. Hence, PDS is potentially suitable for the long-term preservation of sporulated coccidian oöcysts as type-specimens for taxonomic purposes. The samples used in this study are now in the care of the Natural History Museum, London, UK. It is recommended that they be monitored in like manner, by suitably qualified scientists, at intervals of about 5 years to assess their state of preservation and the recoverability of DNA. Enough material is available to monitor it until it is at least 100 years old.
authors.affiliation: Coxitec Consulting, Hertfordshire, UK. ray.coxitec@tesco.net
authors.forename: R B
authors.initials: RB
authors.lastname: Williams
authors.forename: P
authors.initials: P
authors.lastname: Thebo
authors.forename: R N
authors.initials: RN
authors.lastname: Marshall
authors.forename: J A
authors.initials: JA
authors.lastname: Marshall
mesh_terms: D000818:Animals; D002645:Chickens / Q000469:parasitology*; D016054:DNA, Protozoan / Q000235:genetics*; D004539:Eimeria / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D005404:Fixatives / Q000494:pharmacology; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction; D011192:Potassium Dichromate / Q000494:pharmacology; D011309:Preservation, Biological / Q000379:methods*; D033761:Spores, Protozoan / Q000235:genetics*; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan; D005404:Fixatives; D011192:Potassium Dichromate
references.citation: Adv Pharmacol Chemother. 1973;11:221-93
references.pmid: 4591806
references.citation: Avian Pathol. 2009 Oct;38(5):359-66
references.pmid: 19937523
references.citation: Z Parasitenkd. 1979 Feb 28;58(2):115-39
references.pmid: 107666
references.citation: J Parasitol. 1978 Feb;64(1):163-4
references.pmid: 75264
references.citation: J Parasitol. 1988 Aug;74(4):519-23
references.pmid: 3294365
references.citation: Parasitol Today. 1986 Nov;2(11):314-6
references.pmid: 15462747
references.citation: J Parasitol. 1997 Apr;83(2):333-6
references.pmid: 9105325
references.citation: J Parasitol. 1991 Feb;77(1):52-7
references.pmid: 1899451
references.citation: Vet Parasitol. 2007 May 15;146(1-2):35-45
references.pmid: 17386979
references.citation: J Parasitol. 1985 Dec;71(6):808-14
references.pmid: 4093813
references.citation: Avian Dis. 2002 Oct-Dec;46(4):775-802
references.pmid: 12495038
references.citation: J Parasitol. 1988 Aug;74(4):737-9
references.pmid: 3397838
references.citation: Int J Parasitol. 2001 Aug;31(10):1056-69
references.pmid: 11429169
references.citation: Parasitology. 1978 Aug;77(1):27-31
references.pmid: 756523