Genetic diversity amongst isolates of Neospora caninum, and the development of a multiplex assay for the detection of distinct strains

Abstract

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.

Fig. 1

PCR amplification of (A) Tand-4 and (B) Tand-15. (A) Lanes: M = marker, 1 = NC1, 2 = NC-Beef, 3 = BPA1, 4 = NC-SweB1, 5 = NC-Nowra, 6 = NC-Liverpool. (B) Lanes: M = marker, 1 = BPA1, 2 = NC-SweB1, 3 = NC-Illinois, 4 = NC-Nowra, 5 = NC-Liverpool. The pictures show the outcome of a preliminary screen for diversity amongst isolates in their repetitive DNA.

Fig. 2

PCR amplification of repetitive sequences from different N. caninum isolates. (A) Tand-12; (B) Tand-13; (C) Tand-3. Lanes M = marker (given in bp), R = control, V = Vero, 1 = T. gondii, 2 = NC-Liverpool, 3 = NC-Nowra, 4 = NC-SweB1, 5 = BPA1, 6 = BPA6, 7 = NC-Beef, 8 = JAP1, 9 = WA-K9, 10 = NC-Bahia, 11 = NC-LivB1, 12 = NC-LivB2, 13 = NC-GER1, 14 = NC-GER2, 15 = NC-GER3, 16 = NC-GER4, 17 = NC-GER5, 18 = NC-GER6, 19 = NC-GER8, 20 = NC-GER9.

Fig. 3

Multiplex PCR was performed for the three markers: Tand-3 Tand-12 and Tand-13. Lanes: M = marker (given in bp), R = control, V = Vero, 1 = T. gondii, 2 = Hammondia heydorni, 3 = NC-Liverpool, 4 = NC-Nowra, 5 = NC-SweB1, 6 = BPA1, 7 = BPA6, 8 = NC-Beef, 9 = JAP1, 10 = WA-K9, 11 = NC-Bahia, 12 = NC-LivB1, 13 = NC-LivB2, 14 = NC-GER1, 15 = NC-GER2, 16 = NC-GER3, 17 = NC-GER4, 18 = NC-GER5, 19 = NC-GER6, 20 = NC-GER8, 21 = NC-GER9, 22 = NC-Illinois, 23 = NC1.

Fig. 4

Sensitivity of multiplex PCR analysis assessed using different amounts of purified NC-Nowra DNA as template.

Fig. 5

Multiplex PCR of DNA extracted from pup brains showing that NC-Liverpool isolate is present in the brains. Lanes: M = marker (given in bp), R = control, Now = NC-Nowra, Liv = NC-Liverpool, 94–97 = negative control of mouse brain, 3–21 = mouse brains from a challenge experiment infected with NC-Liverpool.

Fig. 6

Multiplex PCR of DNA extracted from dog serum that was antibody positive to N. caninum. Lanes: M = marker (given in bp), 1 = DNA from NC-Nowra, 2 = DNA from dog serum.