Abortion is economically important in many types of ruminants in Europe, North America, Africa, and Asia. C. abortus gives rise to reproductive disorders, inflammation of the epididymis, pneumonia, arthritis and conjunctivitis. It is even found in the feces of healthy sheep and goats and could also be a zoonotic risk for pregnant women [21, 25, 28].
In several studies, molecular techniques, such as various PCR assays and real-time PCR based on different goal genes (ompA, IGS-SrRNA, 16SrRNA, pomp90-3, pomp90-4, and 16s-23s spacer region) have been used for the detection or phylogenic analysis of C. abortus strains in the aborted fetuses of ewes [22, 29, 30, 31]. Certainly, these methods have different sensitivity and specificity.
The role of Chlamydiosis in different types of animals in Iran, particularly in the 2 studied provinces, has been investigated by serologic and molecular tests; however, due to the complications of the culture method and common clinical signs, isolation has never been the focal point of research. Therefore, isolation and genotyping of C. abortus were conducted by the present research in Iran. Undoubtedly, this research is the first attempt to carry out in-depth studies in this context.
In the present study, for the primary detection of C. abortus in the infected samples, real-time PCR was used. By this method, the probability of an error in the differentiation of C. psittaci and C. abortus or missing the coinfection with 2 species is the least. Based on the results, the Chlamydial infection of ruminants in the present research was 34.1% whereas the C. abortus infection revealed to be 100%, which are considerably high percentages. It is worth noting that in spite of the equal number of collected samples in 2 studied provinces, the detected positive samples in Charmahal/Bakhtiari outnumbered those in Khuzestan. This could be related to more traditional training of ruminants in Charmahal/Bakhtiari compared to Khuzestan.
It is necessary to show the relationship between pathogenic and non-pathogenic strains as well as the distribution patterns of the pathogenic strains in the region due to being regarded as determining factors to control pathogenic strains. Pathogenic typing can assist us in understanding the pathogenic relationship between the prevalent types and their sources (human or animal) and the differentiation of current and new infections [32].
The efficacy of the MLVA method has been confirmed by several researchers for the purpose of typing different microorganisms. Laroucau et al. (2009) used 8 selected loci in 9 standard strains of C. psittaci in birds for typing 150 clinical C. psittaci isolates with different hosts in various areas. They stated that this method has a higher diagnostic value and more sensitivity compared with serotyping by ompA gene sequencing. Genotyping of C. abortus strains based on five selected loci (ChlaAb_457, ChlaAb_581, ChlaAb_620, ChlaAb_914, and ChlaAb_300) has been shown to be appropriate for clinical samples [6].
Li et al. (2015), studied 135 samples (9 aborted fetuses, 126 vaginal swabs) of yak papulation in China. C. abortus infection was reported in 9 aborted fetuses (100%) and 30 vaginal swabs (23.81%). MLVA typing of 4 isolated strains in this study was grouped in MT2 [33].
Also, Malal and Turkyilmas in Turkey studied the material samples of abortion in 267 cattle, 380 sheep, 70 goats and 13 water buffaloes. C. abortus infection was reported in 11.9% of the studied samples and as a result of MLVA typing, MT2 was found as the dominant genotype (93.1%). Certainly, genotype 3, 4 and 5 were involved in the infections [34].
In the present research, a study on genetic linkage among C. abortus strains in 2 provinces of Iran was carried out for the first time, using MLVA. According to our findings, there was roughly 80–100% similarity between sheep and bovine C. abortus strains in the 2 studied regions, respectively. The MLVA typing method, based on the analysis of 5 VNTR loci, enabled the clustering of 40 C. abortus strains into 8 genotypes; this showed genotyping diversity among C. abortus strains which included 8 different MLVA types (MT1-8). Variation of MTs in Charmahal/Bakhtiari (MT1- MT8) was more than that of Khuzestan (MT1, MT3) province, which could be related to different quantities of positive samples in each area. There was a close genetic linkage among the strains from Khuzestan. Furthermore, the occurrence of common clones from Khuzestan and Chaharmahal/Bakhtiari indicated a genetic similarity in C. abortus strains from these regions and between the sheep and cattle strains. This provides evidence for the transmission risk of C. abortus ruminant infection from these areas. In the same vein, Seth-Smith et al. (2017) reported that the diversity within C. abortus as compared to other species is much lower; moreover, the genome of C. abortus is highly stable [13]. Therefore, suitable strategies should be employed to curb the possible spread of infection due to C. abortus in sheep and cattle in the studied regions. Due to the proximity of these 2 provinces and the migration of animals between them, these findings were expected relatively.
In the current research, MT1 was reported as the dominant type but in another research regarding C. abortus in Asia (Turkey and China), MT2 turned out to be dominant. Considering the common border between Iran and Turkey and also the probability of animal interchange, augmented similarity in MLVA type is expectable. Any investigation regarding C. abortus genotyping in areas where Turkey shares border with West Azarbayejan province can shed light on such variations. The exact reason behind different predominant C. abortus MLVA type in areas with no common borderline such as China or any European country could be due to various management and hygienic factors.
Siarkou et al. (2015) reported 7 types in the MLVA genotyping of 94 ruminant C. abortus isolates, with the highest repetition variation occurring in MT2 [35]. The correlation between repetitive locus number and the virulence of strains was mentioned in the Laouracu et al. (2008) study [20]. In the present research, 3 strains were demonstrated with 4 repetitions of 5 loci which are more acute compared with other strains. A survey of the repetitive profile (5 studied loci) in 40 isolates showed that the ChlaAb_457 and ChlaAb_620 loci appeared in 97.5% and 0% of the isolates, respectively.
The collection of samples was carried out over a period of 3 years (2014-2016); therefore, the time difference can also be investigated in the present research. The highest and the lowest variations were observed in MT1 and MT7, respectively. The isolates in the other types (MT2, MT3, MT4, MT5, and MT6) were a combination across 3 years which could be indicative of the genetic relationship between these types.