Analysis of Toxoplasma gondii clonal type-specific ... · PDF fileAnalysis of Toxoplasma...
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Analysis of Toxoplasma gondii clonal type-specific antibody reactions in
experimentally infected turkeys and chickens by peptide microarray
P. Maksimov 1, W. Basso 2, J. Zerweck 3, Mike Schutkowski 3, A. Maksimov 1,
F. J. Conraths 1, G. Schares 1
1 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, D-17493, Greifswald-
Insel Riems, Germany2 Institute of Parasitology, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland
3 JPT Peptide Technologies GmbH, Volmerstrasse 5, D-12489 Berlin, Germany
Toxoplasmosis occurs in humans and
animals worldwide.
T. gondii may cause severe clinical disease.
There are indications of correlations between
clinical manifestations and the clonal type
of T. gondii humans are infected with.
Toxoplasma gondii has a clonal population
structure. In North America and Europe three
clonal types (I, II, III) dominate with clonal
type II being the most prevalent one.
There is limited information about the ability
of turkeys and chickens to develop clonal
type specific antibody response.
Aims of this study
Development of a serotyping test
based on peptide microarray using
reference sera from experimentally
infected turkeys and chickens.
Analyse the ability of turkeys and
chickens to develop a T. gondii clonal
type-specific antibody response (IgY).
Material and Methods
Peptides
101 peptides representing sequences of T. gondii clonal type specific
antigenic sites were analysed in peptide microarray.
The peptide panel consisted of peptides presenting single clonal
type specific polymorphisms (I [n = 27], II [n = 29] and III [n = 21]) as
well as common polymorphisms for two clonal types simultaneously
(I/II [n = 6], I/III [n = 12], II/III [n = 6]).
Reference sera
Reference sera: 120 sera from experimentally infected chickens and
turkeys inoculated with different doses of T. gondii tachyzoites (104,
103 and 102) collected from isolates representative for types I (RH), II
(ME49) or III (NED) and uninfected controls. The sera were collected
at 0, 2, 5, 7 and 9 weeks post infection (wpi).
Results
All experimentally infected turkeys and chickens seroconverted in the
TgSAG1-ELISA (Maksimov et al., 2011; Schares et al., 2016) (Fig. 1)
After screening the peptides with reference sera from chickens and
turkeys, 30 and 37 peptides were identified that showed type specific
reactions with sera collected 2, 5, 7 and 9 wpi as determined by
Receiver Operating Characteristics (ROC) analysis. These peptides
originated from eight T. gondii antigens (ROP1, GRA1, GRA3,
GRA5, GRA6, GRA7, SAG2A, SAG3). In addition, turkey sera
recognized peptides derived from the SAG1 T. gondii antigen.
With selected peptides it was possible to determine until 7 wpi the T.
gondii clonal type, by which groups of chickens and turkeys had
been infected (Fig. 2). Differences in recognized peptide patterns
were observed between individual animals as well as between
different time points post infection.
Most of the animals recognized peptide patterns, by which at least
the infection with one out of three T. gondii clonal types could be
excluded. At 9 wpi, most of the experimentally infected chickens
(78% [14/18]) and turkeys (78% [14/18]) did no longer react with the
selected peptides (Fig. 2 B, C and D).
Summary
Experimentally infected chickens and turkeys are able to develop
a clonal type specific IgY antibody response.
Serotyping of the infection in individual chickens or turkeys was
only possible when the whole peptide panel was applied.
Serotyping using the selected peptides was only possible in a
certain time period post infection.
A B
Fig. 1 Time course of antibody responses in turkeys (A) and chickens (B) as determined by
the TgSAG1-ELISA. control indicates uninfected groups, I indicates groups infected with T.
gondii tachyzoites collected from isolates representative for type I (RH), II indicates groups
infected with T. gondii tachyzoites collected from isolates representative for type II (ME49)
and III indicates groups infected with T. gondii tachyzoites collected from isolates
representative for type III (NED).
References
1. Maksimov et al., 2011, Veterinary Parasitology 182 (2011) 140– 149
2. Schares et al., 2016, In Press, https://doi.org/10.1016/j.exppara.2016.11.004
Background
Type I infected animals Type II infected animals Type III infected animals
D E F
A B C
Fig. 2 Number of serum-peptide reactions from experimentally infected turkeys (A-C) and
chickens (D-F) stratified by wpi and specificity of recognized peptides. The recognized
peptides were divided into two groups: in expected (+) i.e. peptides with specificity
corresponding with type of infection (peptides derived from type I & I_III are expected to be
recognized by animals infected with type I (A, D); peptides derived from type II are expected
to be recognized by animals infected with type II (B, E); and peptides derived from type III &
I_III are expected to be recognized by animals infected with type III (C, F)) and unexpected
(-), all those peptides derived from plymorphic sites non corresponding with type of
infection.
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