Genotyping of Chlamydia abortus using multiple loci variable number of tandem repeats analysis technique

Abstract

Background: The correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus.

Methods: The aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique.

Results: Forty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of aborted fetuses). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6). Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6).

Conclusion: By this research revealed that C.abortus was responsible for a significant percentage of ruminant abortion in two studied regions. The main MT type was MT1 (32% of aborted fetuses) and also 7 different genotypes were involved in infections. So it is concluded that diversity in C.abortus genotyping is high in two regions.

Keywords: Chlamydia abortus; Genotyping; Iran; MLVA; Ruminants.

Fig. 1

Demonstration of geographical situation of two studied provinces

Fig. 2

Giemsa staining of inoculated egg yolk sac with positive sample after 72 h (ED11). N and arrows demonstrate normal cells and inclusion in the infected cells, respectively

Fig. 3

Morphology of C. abortus inclusion bodies (arrows) in L929 cells × 100. a & b; Giemsa staining of non- trypsinated infected cells; the long and short arrows show the normal and infected cells with inclusion, respectively. C & d; Giemsa staining of inclusion bodies (arrows) in trypsinated C. abortus infected cells

Fig. 4

(A) PCR results with Chlamydiales specific primers cIGS-1f/ cIGS-1r in agarose gel N– negative control; M - molecular mass standard 100 bp ladder; P– positive control (352 bp); lines 1 and 2- tested samples. (B) PCR results with C. abortus pomp90–3 specific primers. N– negative control; P – positive control (222 bp); Lines 1–3 tested samples; M - molecular mass standard 100 bp ladder.

Fig. 5

Indirect fluorescent antibody staining of non trypsinated C. abortus infected cells; Inclusion and infected cells are demonstrated with arrow

Fig. 6

MLVA profile of C. abortus in 2 studied samples. M - Molecular mass standard 100 bp ladder; N-negative control; P- C. abortus S26/3 as positive control; lines 1 and 2- tested samples .

Fig. 7

Dendrogram of C. abortus based on MLVA analysis. Results are compared using the Dice method and clustered by UPGMA.