Genetic diversity amongst isolates of Neospora caninum, and the
development of a multiplex assay for the detection of distinct strains
, M.P. Reichel
, A. Ivens
, J.T. Ellis
Department of Medical and Molecular Biosciences, University of Technology, Sydney, P.O. Box 123,
Broadway, New South Wales 2007, Australia
Wellcome Trust Sanger Centre, Hinxton Hall, Hinxton, Cambs, UK
Received 7 January 2009
Accepted 29 January 2009
Available online 11 February 2009
Infection with Neospora caninum is regarded as a signiﬁcant cause of abortion in cattle. Despite the
economic impact of this infection, relatively little is known about the biology of this parasite. In this
study, mini and microsatellite DNAs were detected in the genome of N. caninum and eight loci were
identiﬁed that each contained repetitive DNA which was polymorphic among different isolates of this
parasite. A multiplex PCR assay was developed for the detection of genetic variation within N. caninum
based on length polymorphism associated with three different repetitive markers. The utility of the
multiplex PCR was demonstrated in that it was able to distinguish amongst strains of N. caninum used as
either vaccine or challenge strains in animal vaccination experiments and that it could genotype N.
caninum associated with naturally acquired infections of animals. The multiplex PCR is simple, rapid,
informative and sensitive and should provide a valuable tool for further studies on the epidemiology of N.
caninum in different host species.
Ó 2009 Elsevier Ltd. All rights reserved.
Neospora caninum is a cyst-forming apicomplexan parasite,
which can invade many different cell types and tissues. It causes
stillbirth and abortion in cattle and neuromuscular disorders in
dogs, although infections have been reported in several other host
species [1–4]. Neosporosis is now considered to be the main cause
of infectious bovine abortion around the world [3,5,6].
There are marked differences in pathogenicity (virulence) and
growth rates amongst isolates of N. caninum [7–9]. For example,
NC-Liverpool causes a substantial inﬂammatory response in the
central nervous system of the mouse, leading to disease, whereas
NC-Nowra does not . Therefore, the biological and genetic
diversity among N. caninum isolates could impact on the patho-
genesis of neosporosis in both cattle and dogs and might also be an
important consideration in the design and application of vaccines
Diversity in biological characteristics may also have consider-
able implications for our understanding of the epidemiology of
neosporosis. Detailed information about the genetic diversity
among different geographical isolates of N. caninum, however, is
scant. Genetic diversity among isolates of N. caninum has been
detected using ribosomal DNA sequencing; the analysis of inverted
repetitive DNA and microsatellite repetitive DNA [7,8,11,12].To
understand genetic diversity within N. caninum and the contribu-
tion of these genetic differences to the heterogeneity in disease
manifestation as well as transmission patterns for developing new
strategies for vaccination or diagnosis, the detection of genetic
variation (‘‘strain typing’’) using suitable markers is essential. Any
such markers should be simple, stable, rapid, reproducible and
Mini and microsatellites are repetitive DNA sequences in the
genomes of eukaryotic organisms, containing tandem repeats of
a DNA motif. They are highly polymorphic in sequence and length
[15,16]. Microsatellites are short, tandem repeats (2–6 bases long),
whereas minisatellites have longer repeat units (8–100) [17,18].
Recently, a study of microsatellite markers described genetic
diversity amongst nine isolates of N. caninum , and a number
of polymorphisms were detected. No relationship was found
between the organisation of the nucleotide repeat and the
degree of polymorphism. Multilocus analysis revealed that each
of the nine isolates displayed a unique proﬁle and revealed no
association between the DNA proﬁle and host or geographical
Corresponding author. Tel.: þ61 2 9514 4161; fax: þ61 2 95148206.
E-mail address: email@example.com (J.T. Ellis).
Present address: Fios Genomics Ltd, ETTC, King’s Buildings, Edinburgh, EH9
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Molecular and Cellular Probes
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0890-8508/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
Molecular and Cellular Probes 23 (2009) 132–139