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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