Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin–trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor–I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.
References
1.
AndreeKBGresoviacSJHedrickRP: 1997, Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of Myxobolus cerebralis, the causative agent of whirling disease in salmonid fish. J Eukaryot Microbiol44:208–215.
2.
AndreeKBHedrickRPMacConnellE: 2002, A review of the approaches to detect Myxobolus cerebralis, the cause of salmonid whirling disease. Am Fish Soc Symp29:197–211.
3.
AndreeKBMacConnellEHedrickRP: 1998, A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss. Dis Aquat Org34:145–154.
4.
AntonioDBAndreeKBMcDowellTSHedrickRP: 1998, Detection of Myxobolus cerebralis in rainbow trout and oligochaete tissues by using a non-radioactive in situ hybridization (ISH) protocol. J Aquat Anim Health10:338–347.
5.
BaldwinTJMyklebustKA: 2002, Validation of a single round polymerase chain reaction assay for identification of Myxobolus cerebralis myxospores. Dis Aquat Org49:185–190.
6.
BaldwinTJVincentERSilflowRMStanekD: 2000, Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions. J Vet Diagn Invest12:312–321.
7.
BeauchampKAGayMKelleyGO: 2002, Prevalence and susceptibility of infection to Myxobolus cerebralis, and genetic differences among populations of Tubifex tubifex. Dis Aquat Organ51:113–121.
8.
ChiangPSongWWuK: 1996, Use of fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance. Genome Res6:1013–1026.
9.
El-MatbouliMHoffmannRW: 1991, Prevention of experimentally induced whirling disease in rainbow trout Oncoryhnchus mykiss by Fumagillin. Dis Aquat Org10:109–113.
10.
El-MatbouliMHoffmannRW: 1998, Light and electron microscopic study on the chronological development of Myxobolus cerebralis in Tubifex tubifex to the actinosporean stage triactinomyxon. Int J Parasitol28:195–217.
11.
El-MatbouliMHoffmannRWMandokC: 1995, Light and electron microscopic observations on the route of the triactinomyxon-sporoplasm of Myxobolus cerebralis from epidermis into rainbow trout cartilage. J Fish Biol46:919–935.
12.
GutMLeuteneggerCMHuderJBLutzH: 1999, FCoV quantification using the TaqMan® fluorogenic real-time detection system. J Virol Methods77:37–46.
13.
HallidayMM: 1976, The biology of Myxosoma cerebralis, the causative agent of whirling disease of salmonids. J Fish Biol9:339–357.
14.
HedrickRP: 1998, Relationships of the host, pathogen, and environment: implications for diseases of cultured and wild fish populations. J Aquat Anim Health10:107–111.
15.
HedrickRPEl-MatbouliMAdkisonMAMacConnellE: 1998, Whirling disease: re-emergence among wild trout. Immunol Rev166:365–376.
16.
HedrickRPMcDowellTSGayM: 1999, Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease. Dis Aquat Org37:173–183.
17.
HedrickRPMcDowellTSMukkatiraK: 1999, Susceptibility of selected inland salmonids to experimentally induced infections with Myxobolus cerebralis the causative agent of whirling disease. J Aquat Anim Health11:330–339.
18.
HedrickRPMcDowellTSMukkatiraK: 2001, Susceptibility of three species of anadromous salmonids to experimentally induced infections with Myxobolus cerebralis, the causative agent of whirling disease. J Aquat Anim Health13:43–50.
19.
HedrickRPWishovskyAModinJCTothRJ: 1991, Three myxosporeans found in the cranial and branchial tissues of rainbow trout in California. J Aquat Anim Health3:55–62.
20.
HoffmanGLPutzRE: 1969, Host susceptibility and the effect of aging, freezing, heat, and chemicals on spores of Myxosoma cerebralis. Prog Fish Cult31:35–37.
21.
HollandPMAbramsonRDWatsonRGelfandDH: 1991, Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA88:7276–7280.
22.
LeuteneggerCMHigginsJMatthewsTB: 2001, Realtime taqman PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus DNA. AIDS Res Hum Retroviruses17:243–251.
23.
LeuteneggerCMKleinDHofmann-LehmannR: 1999, Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan® fluorgenic realtime detection system. J Virol Methods78:105–116.
24.
LeuteneggerCMMislinCNSigristB: 1999, Quantitative real-time PCR for the measurement of feline cytokine mRNA. Vet Immunol Immunopathol71:291–305.
25.
LomJDykováI: 1992, Protozoan parasites of fishes. Elsevier, New York, NY.
26.
LorzHVAmandiA: 1994, VI. Whirling disease of salmonid. In: Suggested procedures for the detection and identification of certain finfish and shellfish pathogens, ed. ThoesenJC, 4th ed., pp. 1–7. Am Fish Soc, Fish Health Section, Bethesda, MD.
27.
MacConnellEVincentER: 2002, The effects of Myxobolus cerebralis on the salmonid host. Am Fish Soc Symp29:95–107.
28.
MaicasSAdamACPolainaJ: 2000, The ribosomal DNA of the Zygomycete Mucor miehei. Curr Genet37:412–419.
29.
MarkiwME: 1989, Salmonid whirling disease: myxosporean and actinosporean stages cross-react in a direct fluorescent antibody test. J Fish Dis12:137–141.
30.
MarkiwME: 1991, Whirling disease: earliest susceptible age of rainbow trout to the triactinomyxid of Myxobolus cerebralis. Aquaculture92:1–6.
31.
MarkiwME: 1992, Experimentally induced whirling disease. I. Dose response of fry and adults of rainbow trout exposed to the triactinomyxon stage of Myxobolus cerebralis. J Aquat Anim Health4:40–43.
32.
MarkiwMEWolfK: 1974, Myxosoma cerebralis: isolation and concentration from fish skeletal elements-sequential enzymatic digestions and purification by differential centrifugation. J Fish Res Bd Can32:15–20.
33.
MarkiwMEWolfK: 1983, Myxosoma cerebralis (Myxozoa: Myxosporea) etiologic agent of salmonid whirling disease requires tubificid worms (Annelida: Oligochaetea) in its life cycle. J Protozool30:561–564.
34.
NehringRBWalkerPG: 1996, Whirling disease in the wild: the new reality in the intermountain west. Fisheries (Bethesda)21:28–32.
35.
O'GrodnickJJ: 1975, Whirling disease Myxosoma cerebralis spore concentration using the continuous plankton centrifuge. J Wildl Dis11:54–57.
36.
O'GrodnickJJ: 1979, Susceptibility of various salmonids to whirling disease (Myxobolus cerebralis). Trans Am Fish Soc108:187–190.
37.
PusterlaNHuderJBLeuteneggerCM: 1999, Quantitative realtime PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks. J Clin Microbiol37:1329–1331.
38.
RoseJDMarrsGSLewisCSchislerG: 2000, Whirling disease behavior, and its relation to pathology of brain stem, and spinal cord in rainbow trout. J Aqua Anim Health12:107–108.
SchislerGJBergersenEPWalkerPG: 2001, Comparison of single-round polymerase chain reaction (PCR) methods for detection of Myxobolus cerebralis. Dis Aquat Org45:109–114.
41.
SollidSSLorzHVStevensDGBartholomewJL: 2002, Relative susceptibility of selected Deschutes River, Oregon, salmonid species to experimentally induced infection by Myxobolus cerebralis. Am Fish Soc Symp29:117–124.
42.
ThompsonKGNehringRBBowdenDCWygantT: 1999, Field exposures of seven species or subspecies of salmonids to Myxobolus cerebralis in the Colorado River, Middle Park, Colorado. J Aquat Anim Health11:312–329.
43.
ThompsonKGNehringRBBowdenDCWygantT: 2002, Response of rainbow trout Oncorhynchus mykiss to exposure to Myxobolus cerebralis above and below a point source of infectivity in the upper Colorado River. Dis Aquat Org49:171–178.
44.
VincentER: 1996, Whirling disease and wild trout: the Montana experience. Fisheries (Bethesda)21:32–34.
45.
VincentER: 2002, Relative susceptibility of various salmonids to whirling disease with emphasis on rainbow and cutthroat trout. Am Fish Soc Symp29:109–115.
46.
WagnerEJ: 2002, Whirling disease prevention, control, and management: a review. Am Fish Soc Symp29:217–225.
47.
WhiteMMMcLarenIA: 2000, Copepod development rates in relation to genome size and 18S rDNA copy number. Genome43:750–755.
48.
WolfKMarkiwME: 1984, Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science225:1449–1452.
49.
WolfKQuimbyMC: 1962, Established eurythermic line of fish cell in vitro. Science136:1065–1066.
