Parasite load in guinea pig foetus with real time PCR after maternofoetal transmission oftoxoplasma gondii

Abstract

Summary:Parasite loads o f different tissues w ere assessed in guinea pig foetus after maternal infection. Twelve fem ale guinea pigs w ere infected w ith 1 0 0 cysts o f the 7 6 K strain o f Toxoplasma g o n d ii by the oral route. Inoculation w as performed 2 0 ± 5 days (G 2 0) or 4 0 ± 5 days (G 4 0) after the beginning o f gestation. G estational a g e w as determined by progesterone assay. M aternal and foetal organ samples w ere taken 6 0 days after the beginning of gestation. Parasite loads (from placenta, am niotic fluid (AF), cord blood (CB), foetal brain, liver, lung and spleen) w ere assessed by a real-time PCR quantification using fluorescence resonance energy transfer (FRET) hybridization probes on the Light C ycler®. C ongenital transmission w as proven by the presence of parasites in blood or tissue samples o f the foetus in 8 4 .6 % ( 1 1 /1 3 ) and 1 0 0 % ( 1 6 /1 6 ) o f cases after inoculation on G 2 0 and G 4 0 , respectively. The quantitative analysis o f our results after inoculation at G 2 0 and G 4 0 has allo w e d us to determinate the positive parasitic loads as a function o f the origin o f the sample and the period of inoculation. The parasite loads expressed as log (p a ra site /g ) w ere low in AF and CB samples: 1 .4 9 ± 0 .5 0 and 1 .0 5 ± 0 .1 0 at G 2 0 and 1.21 ± 0 .3 6 and 1 .2 0 ± 0 .4 2 at G 4 0 respectively. In contrast the placenta and the different foetal tissues had higher parasite burdens: 2 .8 9 ± 0 .5 4 to 5 .3 0 ± 0 .5 1 at G 2 0 and 2 .8 1 ± 0 .7 1 to 3 .6 5 ± 0 .5 9 at G 4 0 . All the placentae w ere positive for parasites even in the tw o cases with no proven transmission. Real time quantitative PCR using the hybridization probe w as a very sensitive and reproducible technique to study the kinetics o f congenital toxoplasmosis in the guinea pig model w ich is close to that o f humans.KEY WORDS : congenital toxoplasmosis, parasite load, guinea-pig, real-time PCR.R ésu m é : D é t e r m in a t io n p a r PCR q u a n t it a t iv e en t e m p s r é el d e la c h a r g e t o x o p l a s m iq u e d e t is s u s d e f oe t u s d e c o b a y e s a p r è s in f e s t a t io n m a t er n e l l e Les charges toxoplasmiques de différents tissus de foetus de cobaye ont été déterminées après infestation maternelle. Douze cobayes gestantes ont été infestées avec 100 kystes de la souche 7 6 K pa r voie orale. C ette infestation a eu lieu après le début de la gestation soit à 2 0 jours (± 5 ) (G20), soit à 4 0 jours (± 5 ) (G 4 0 ) de gestation. Le prélèvement des organes foetaux a été effectué à 6 0 jours de gestation (G 6 0 ) soit cinq jours avant terme. Les charges parasitaires (Placenta, liquide amniotique (L A ), sang de cordon (SC), foie, cerveau, poumon, et rate) ont été déterminées pa r PCR quantitative en temps réel (technique Sonde-sonde) sur Light-Cycler®. L'analyse qualitative de nos résultats nous a permis d'affirmer une transmission maternofoe tale (organes foetaux e t/o u sang de INTRODUCTION_____ ______

A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation

“…This DNA element is repeated 35 times in the genome of the parasite, and the number of repeats provides optimal detection of T. gondii DNA by PCR (18). Recently, a 529 bp element that is repeated 200–300 times in the genome of T. gondii has been described (9, 19–23). It has been proposed that a PCR target containing more repeats than the B1 gene may increase the analytical sensitivity (24–28).…”

Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis

“…A variety of real-time PCR assays and PCR targets have been developed for the rapid and sensitive detection of T. gondii [4][5][6], including the B1 gene, which occurs in 35 copies in the parasite genome [7][8][9][10][11][12][13]. A further repeat element of unknown function, which occurs in 200-300 copies, has recently been described in T. gondii [14].…”

Effects of Neospora caninum Infection at Mid-Gestation on Placenta in a Pregnant Mouse Model

“…In previous reports, in mice infected at late mid-gestation (day 10 of gestation), N. caninum tachyzoites were identified by immunohistochemistry in the placenta and fetus on days 6-8 PI (Long and Baszler, 1996;Rettigner et al, 2004). In a guinea pig model of Toxoplasma gondii infection, the placental parasite load was also time-dependent, and a higher parasite burden was found in the placenta compared to the fetuses in the early stages of the infection (Flori et al, 2003). Taken together, these findings may indicate that the parasite is mainly multiplying in the placenta during the initial infection.…”