DIFFERENTIATION OF DORSAL-SPINED ELAPHOSTRONGYLINE LARVAE BY POLYMERASE CHAIN REACTION AMPLIFICATION OF ITS-2 of rDNA

Gajadhar, Alvin A.; Steeves-Gurnsey, Teresa; Kendall, Jane C.; Lankester, Murray W.; Stéen, Margareta

doi:10.7589/0090-3558-36.4.713

Cited by 37 publications

(40 citation statements)

References 16 publications

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“…Differences in the second internal transcribed spacer (ITS-2) of ribosomal DNA have been used to identify many nematode parasites (Gasser and Monti, 1997;Criscione et al, 2005), including specimens of DSLs among elaphostrongylines and other protostrongyles (Gajadhar et al, 2000). Restriction digestion of this locus, however, has proven to be equivocal, and it has now been demonstrated that direct sequence comparisons are required to differentiate among species of Parelaphostrongylus if the ITS-2 is the basis for identification .…”

Section: Introductionmentioning

confidence: 99%

Parelaphostrongylus Odocoilei in Columbian Black-Tailed Deer From Oregon

Mortenson

Abrams

Rosenthal

et al. 2006

Journal of Wildlife Diseases

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ABSTRACT:Documenting the occurrence of Parelaphostrongylus odocoilei has historically relied on the morphological examination of adult worms collected from the skeletal muscle of definitive hosts, including deer. Recent advances in the knowledge of protostrongylid genetic sequences now permit larvae to be identified. Dorsal-spined larvae (DSLs) collected in 2003-2004 from the lung and feces of six Columbian black-tailed deer (Odocoileus hemionus columbianus) from Oregon were characterized genetically. The sequences from unknown DSLs were compared to those from morphologically validated adults and larvae of P. odocoilei at both the second internal transcribed spacer (ITS-2) of ribosomal DNA and the mitochondrial cytochrome oxidase II gene. We provide the first unequivocal identification of P. odocoilei in Columbian black-tailed deer from Oregon. The broader geographic distribution, prevalence, and pathology of P. odocoilei are not known in populations of Oregon deer.

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Section: Introductionmentioning

confidence: 99%

Parelaphostrongylus Odocoilei in Columbian Black-Tailed Deer From Oregon

Mortenson

Abrams

Rosenthal

et al. 2006

Journal of Wildlife Diseases

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“…The second internal transcribed spacer (ITS-2) of the ribosomal (r) DNA was used as the target to define speciesspecific markers. However, in the study by Gajadhar et al, 5 PCR analyses involving larval samples were based on the amplification of DNA prepared from pools of 25 L 1 s because of the difficulty in isolating sufficient and pure DNA template from individual L 1 s, which vary in size from 308 to 490 m long by 16 to 24 m wide. 13 Adequate recovery of gDNA from individual elaphostrongyline larvae has been a major constraint in the development of reliable PCR assays for the diagnosis of infection.…”

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confidence: 99%

“…13,14 Development of diagnostic techniques for the accurate identification of individual elaphostrongyline larvae is important for epidemiological studies and the control of these parasites because they differ in their pathogenicity, host specificity, and geographic distribution. 13 A recent study by Gajadhar et al 5 demonstrated the value of polymerase chain reaction (PCR)-based techniques for the identification of elaphostrongyline nematodes to the species level. The second internal transcribed spacer (ITS-2) of the ribosomal (r) DNA was used as the target to define speciesspecific markers.…”

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confidence: 99%

A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

et al. 2005

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Abstract. This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L 1 ) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L 1 s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L 1 s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L 1 s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.Key words: Diagnosis; DNA extraction; Elaphostrongylus; Parelaphostrongylus; PCR.Elaphostrongyline nematodes (Elaphostrongylus spp. and Parelaphostrongylus spp.) occur in the central nervous system and/or skeletal muscles of cervids and can produce verminous pneumonia and, in some cases, severe neurologic disease. 13 Four of the 6 species of elaphostrongyline, Parelaphostrongylus tenuis, Parelaphostrongylus andersoni, Parelaphostrongylus odocoilei, and Elaphostrongylus rangiferi, occur in North America. 13 Elaphostrongylus rangiferi also occurs in northern Fennoscandinavia and Russia. 13 Elaphostrongylus alces occurs in Fennoscandinavia, whereas Elaphostrongylus cervi occurs in Eurasia and New Zealand. 13 Elaphostrongylus rangiferi was introduced to the island of Newfoundland with reindeer (Rangifer tarandus tarandus) from Norway in 1908, 14 where it subsequently spread to infect woodland caribou (R. tarandus caribou) and moose (Alces alces). 15 Woodland caribou in Newfoundland are also infected with P. andersoni; however, this parasite is significantly less pathogenic than is E. rangiferi in this host species. 13 It is believed that E. rangiferi has not spread with caribou from Newfoundland to mainland Canada; however, this needs to be confirmed. 4 The first-stage larvae (L 1 ) of the elaphostrongylines and of several other genera (e.g., Varestrongylus, Muellerius, and Umingmakstrongylus) within the family Protostrongylidae have a characteristic dorsal spine on their tail. 2 The L 1 s of Muellerius and Varestrongylus can generally be distinguished from the elaphostrongyline L 1 s by their shorter length, 9 whereas Umingmakstrongylus occurs only in muskoxen (Ovibos moschatus). 12 Diagnosis of elaphostrongyline infection in cervids is often based on the detection of L 1 s in feces 4,16 ; however, the larvae cannot be identified to the species or genus level because of their morphological and morphometric similarities. 3,4,13 Sometimes the identities of L 1 s are...

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“…Infections of P. tenuis in WTD can be identified by detecting dorsal-spined larvae in feces followed by polymerase chain reaction (PCR) assay (Gajadhar et al, 2000). However, this method is of little value for detecting infected moose since few ever pass larvae.…”

Section: Introductionmentioning

confidence: 99%

Detection of Anti-Parelaphostrongylus Tenuis Antibodies in Experimentally Infected and Free-Ranging Moose (Alces Alces)

Ogunremi

Lankester

Dergousoff

et al. 2002

Journal of Wildlife Diseases

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Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5-9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3-30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61-243 days post-inoculation (DPI). Five animals tested between 16-25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (rϭ0.98, Pϭ0.02 and rϭ0.95, Pϭ0.04, respectively) among animals harboring adult worms (nϭ4) but not significantly with the number of worms recovered postmortem (peak OD, rϭ0.82, Pϭ0.18; terminal OD, rϭ0.93, Pϭ0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.

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DIFFERENTIATION OF DORSAL-SPINED ELAPHOSTRONGYLINE LARVAE BY POLYMERASE CHAIN REACTION AMPLIFICATION OF ITS-2 of rDNA

Cited by 37 publications

References 16 publications

Parelaphostrongylus Odocoilei in Columbian Black-Tailed Deer From Oregon

Parelaphostrongylus Odocoilei in Columbian Black-Tailed Deer From Oregon

A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

Detection of Anti-Parelaphostrongylus Tenuis Antibodies in Experimentally Infected and Free-Ranging Moose (Alces Alces)

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