Identification of Chlamydophila abortus in Abomasum Content of Aborted Sheep and Goats by PCR

A total of 105 aborted sheep fetuses and 15 aborted goat fetuses were collected from 120 different herds in the lambing seasons 2015-2016 from Aksaray, Konya, Karaman, Isparta, Afyonkarahisar and Niğde provinces. The size of herds was between 40 to 965 animals. C. abortus history of the animals was unknown. Abomasum contents of aborted fetuses were taken aseptically as specimen. The samples were transported to the laboratory under cool condition.

Live microorganisms were inactivated by incubating the samples at 95 °C for 15 min in a thermal block. Genomic DNA was obtained from each sample with Vivantis tissue DNA extraction kit, according to the manufacturer’s protocol. The quantity and quality of DNA was measured at 260 and 280 nm optical density by a spectrophotometer (Nanodrop, ND1000, USA). The extracted DNA samples were kept at -20 °C until used.

C. abortus specific primers were used in PCR assay for amplification of C. abortus DNA (Crealan and Cullough, 2000). The sequence of forward primer was 5′-TGG TAT TCT TGC CGA TGA-3′ and reverse primer was 5′-GAT CGT AAC TGC TTA ATA AAC CG-3. This primer pairs showed a single amplicon that was 479 bp.

The PCR were performed in a total reaction volume of 25 μL containing 2.5 μL of 10 X PCR buffer, 250 μM each of the dNTPs (Fermentas, Vilnius, Lithuania), 1.5 mM MgCl2, 1.25 U of Taq DNA polymerase (Fermentas, Vilnius, Lithuania), 2.5 μL of template DNA, and 1 μM of each primer (IDT, USA). DNA amplification was performed in a thermalcycler (Ependorf, Mastercycler gradient, Germany) with the following steps: 1 X 5 min at 95 °C, 40 X 1 min at 94 °C, 1 min 50 °C, 2 min 72 °C, and a final extension at 72 °C for 7 min. DNA of C. abortus S26/3 strain and nuclease free water was used as positive and negative controls, respectively. The amplified PCR products were analyzed by electrophoresis on 1.5% agarose gel, and the gel was then stained with ethidium bromide (0.5 μg/mL) and photographed.

A total of 120 abomasal contents of the aborted fetuses were collected from the provinces of Central Anatolia of Turkey. Chlamydial DNA was detected in seven (5.8%) of the 120 abomasum content of fetuses by PCR. The 15 aborted goat fetuses were found to be negative for C. abortus. A 479 bp PCR product was amplified from seven (6.7%) out of the 105 aborted sheep fetuses. Among the 7 positive samples, 3, 3, and 1 were collected from Aksaray, Konya and Niğde provinces, respectively. The samples belong to Isparta, Karaman and Afyonkarahisar provinces were found to be negative for C. abortus (Table I).

In this country, limited studies have been conducted for molecular diagnosis of C. abortus in abortion materials. Kalender et al. (2013) examined 71 sheep and goat aborted fetuses by PCR method and reported that 9.8% of the samples were positive for C. abortus. Güler et al. (2006) investigated C. abortus DNA in 172 samples of stomach contents from sheep and goat aborted fetuses and 94 vaginal swab samples and detected it in 6 (3.5%) stomach contents and 7 (7.5%) vaginal swab samples. In the present study, C. abortus DNA was detected in seven (5.8%) of the 120 abomasum content of sheep and goat fetuses by PCR. This result is in harmony with the previous studies. Ovine enzootic abortion is still endemic in Central Regions of Turkey and causes abortion in sheep herds.

In Iran, a total 300 aborted sheep and goat fetuses were investigated by PCR and 11% of samples were found positive for C. abortus (Heidari et al., 2018). In Jordan, Abahneh et al. (2014) examined 66 placental tissues and 15 vaginal swabs collected from aborted ewes by PCR and found that 38 (58 %) placental samples and 13 (87 %) vaginal swabs were positive for chlamydial DNA. Chisu et al. (2013) reported that 3 (7.5%) C. abortus DNA were detected from ovine aborted placental tissues by PCR in Italy. In United Kingdom, 231 ovine aborted tissues (placenta, fetal lung and fetal abomasum content) were investigated for C. abortus by PCR and 73 (31.6%) of samples were found to be positive (Creelan and McCullough, 2000). In our study, the prevalence of ovine enzootic abortion was detected as 5.8%. This rate was lower than result of previous studies that were conducted in Iran, United Kingdom, Jordan, and Italy.

The findings of this study indicated that C. abortus plays an important role in sheep abortion cases in the Central Anatolia. The control and vaccination programs for the fight against C. abortus infection should be determined and implemented in Turkey.

This study was supported by Aksaray University Scientific Research Fund (Project Number: 2015-046).

The authors have declared no conflict of interest.