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Citation Information : Polish Journal of Microbiology. Volume 69, Issue 1, Pages 65-71, DOI: https://doi.org/10.33073/pjm-2020-009
License : (CC-BY-NC-ND 4.0)
Published Online: 28-February-2020
Abortion in small ruminants is a significant problem in Iraq and causes severe economic losses in sheep farms.
Chlamydia abortus (family: Chlamydiaceae) is a non-motile, coccoid, pleomorphic, Gram-negative bacterium. It is an obligate intracellular parasite that causes different diseases in humans and animals (Everett et al. 1999, Madico et al. 2000, Silva et al. 2006).
Ovine chlamydiosis is also called ovine enzootic abortion (OEA) or enzootic abortion of ewes (EAE). It is one of the most significant causal agents of reproductive failure in small ruminants, which is induced by C. abortus (DeGraves et al. 2004, Spičic et al. 2015). The pathogen efficiently colonizes the placental trophoblasts (Borel et al. 2006, Campos-Hernández et al. 2014).
Chlamydiosis causes abortion in sheep and goats in the final 2–3 weeks of pregnancy with the appearance of a stillborn and grossly inflamed placenta (Nietfeld 2001, Walder et al. 2003). Also, C. abortus constitutes a hazard to pregnant women, as many cases have been reported, which were caused by Chlamydia (Pospischil et al. 2002, Walder et al. 2005).
Abortion in the late stages of gestation by Chlamydia results in severe losses in many sheep-raising areas globally, especially where herds are intensely crowded during the parturient period (Halbert 2008, Rodolakis and Mohamad 2010). The disease can lead to the delivery of full-term stillborn lambs. Some of the delivered lambs fail to survive longer than two days. It is also common for an infected ewe to deliver one dead lamb and one or more weak or healthy lambs during multiple births. Infection usually spreads into a new flock by the introduction of infected animals. The disease usually causes a small number of abortions in the first year. In the next year, the rate may increase to infect around 30% of the ewes (Longbottom and Coulter 2003).
Etiologic diagnosis of ovine chlamydiosis depends on bacteriological testing on embryonated chicken eggs and chlamydial DNA amplification by polymerase chain reaction (PCR) (Sachse et al. 2009, Szymanska-Czerwinska et al. 2013).
Infections of C. abortus in sheep have generally been documented serologically in Sulaimani province, in the north of Iraq. However, studies on pathogen isolation and detection by PCR are rare in this region. Therefore, we conducted this study to isolate C. abortus in sheep in Sulaimani province, Iraq using egg-inoculation and PCR molecular techniques. We also conducted a phylogenic analysis to compare our isolate with other Chlamydia species that were deposited in GenBank using the ompA gene as a comparison.
Study area and sample collection. During the birth season of 2017, March to May, abortion in sheep flocks was reported by veterinarians in several regions of Sulaimani province in the northeast of Iraq. The farms had a history of abortion in the previous seasons. The management system of sheep herds in these districts is classical; the animal herds belong to different owners. The sheep are fed indoor on grain, hay, and silage and are left to graze on pasture. Different animal species may graze together on the same pasture or share rams for fertility between herds. Farmers in these areas rear local breed or Arabic (Awasi) sheep.
We collected 30 samples of aborted fetuses from five herds of nonvaccinated sheep in different regions around Sulaimani province. Fifteen samples were from Kalar, ten from Said Sadiq, and five from Chamchamal (Fig. 1). The samples composed of tissue collected by using disposable blades and scissors from recently aborted fetuses (liver, spleen, and lung) and their dams (placental caruncle and cotyledon) that had abortions in the previous 2–4 days. Collected samples were put in plastic containers, labeled, and transferred in a cooled box to the Research Center at the College of Veterinary Medicine/University of Sulaimani for isolation and identification of C. abortus.
Isolation of C. abortus. Tissue samples from cotyledons, placentas, and fetal organs were collected and processed for egg inoculation. Homogenization of the tissues was accomplished by grinding using a sterile pestle and mortar. The homogenized tissues were suspended in the sucrose-phosphate-glutamate (SPG) medium containing sucrose (0.22 mM), K2HPO4 (7.1 mM), L-glutamic acid (4.9 mM), fetal bovine serum (10%), streptomycin (100 μg/ml), gentamicin (50 μg/ml), and nystatin (50 μg/ml) to make a 10% suspension. The suspension was centrifuged at 2000 rpm for 5–10 minutes. After that, the supernatants were collected and aliquoted in small volumes. Part of the supernatant was used directly for the isolation of C. abortus in embryonated eggs, and McCoy cell or the extraction of DNA, and the remainder was stored at –70°C.
For isolation of C. abortus in the yolk sac membranes of chicken eggs, specific-pathogen-free, 6 to 8-day-old embryonated hen’s eggs were inoculated with the tissue suspension of the aborted animals.
After six days of incubation, the vitality of the egg to be inoculated was checked with a candling lamp. Candling (holding an intense light below the egg to observe the embryo in a darkened room) was used to determine and mark the location of the embryo and egg’s air sac. The shell was cleaned with 70% ethanol, and the air sac was marked with a pencil, followed by drilling a small hole into the shell over the top of the air sac. After injecting about 0.2 ml of the inoculums (10% suspension of tissue), the hole was sealed with nail varnish (Soomro et al. 2012). The eggs were incubated at 37°C and candled daily. Eggs that showed embryonic death during 4–10 days after inoculation were kept while the eggs in which the embryo died within 24 hours after inoculation were discarded. Eggs containing dead embryos were kept overnight at 4°C, and the yolk sac membranes were harvested using aseptic techniques in a biosafety cabinet after the rinsing the eggshells with alcohol. The yolk sac was washed two times with pH 7.2 phosphate-buffered saline and cut into small pieces. The pieces were then ground with a pestle and mortar to prepare a cell suspension, as described earlier (Li et al. 2015). The presence of C. abortus was confirmed by staining impression smears with Giemsa. The smears were prepared from highly vascularized areas of the yolk sac membranes of chicken eggs. The smears were then fixed by absolute methanol and stained with Giemsa solution. The stained slides were examined with a light microscope using 1000× magnification for the presence of purple inclusion bodies (Dagnall and Wilsmore 1990).
DNA extraction. The samples were subjected to DNA extraction using a DNA extraction kit (GeNet Bio, South Korea). The procedure was conducted following instructions provided by the manufacturer.
Oligonucleotides and PCR amplification. We used a set of genus-specific primers for discrimination of C. abortus and Brucella abortus from common infectious agents that cause abortion in sheep. The omp-F and omp-R primers were designed from the ompA gene sequence of C. abortus, which encode an amplicon size was 1058 bp and were previously used by Arshi et al. (2011), while B4-F and B5-R primers were designed from the bcsp31 gene sequence of B. abortus. Primers encode an amplicon of 223 bp (Baily et al. 1992). The name, sequence, target gene, the predicted amplified fragment, as well as the melting temperature, are listed in Table I The primers were developed by Macrogen® (South Korea) for our study.
The total DNA was subjected to simplex PCR amplification using PCR PreMix (GeNet Bio, South Korea). The reaction was executed in 0.2 ml PCR tubes. The constituents of the PCR tube were 10 μl master mix, 4 μl DNA, and 1 μl (10 pmol) of each of the forward and reverse primers. The final volume of 20 μl was accomplished by adding 4 μl diethylpyrocarbonate (DEPC)-treated water.
The thermal cycler program was started with denaturation at 95°C for five minutes. After that, 40 cycles of denaturation (95°C for 30 seconds), annealing at 58°C for 30 seconds for C. abortus and 57°C for B. abortus, and extension (72°C for 45 seconds) were run with the final extension at 72°C for five minutes.
The PCR products were examined by loading 7 μl on 1% agarose gel in 1 × Tris/Borate/EDTA (TBE) buffer. The gel was stained with 5 μl Safe dye, and electrophoresis was done using 120 volts for 50 minutes. The Safe-Blue Illuminator/Electrophoresis System was used to visualize the bands of amplicons, which were analyzed based on the pattern of migration by comparing them to a 100 bp DNA ladder.
Sequencing of the ompA gene and phylogenic analysis. The PCR products of the C. abortus ompA gene were sequenced in the Macrogen Sequencing Facility in South Korea. Sequencing was performed several times to verify the identity of each nucleotide, and the gene sequences were presented to GenBank and got the accession number MK643153.
Phylogenic trees were produced using the ompA genes of 75 strains of Chlamydia species (Fig. 3). The sequences were obtained from GenBank and MLST (http://pubmlst.org/chlamydiales/) websites for Chlamydiales. Multiple alignments were done with the Clustal W method (Thompson et al. 1994), and MEGA 7 was implemented to execute a phylogenetic analysis with Neighbor-Joining. The bootstrap measures were determined from 1000 repeats of the original data.
Samples. In the current study, 30 samples were taken from aborted fetuses from different herds of sheep from three districts in Sulaimani. One sample (3.33%) from aborted ewes was found positive for C. abortus both by yolk sac inoculation and PCR (Table II). All the remaining 29 samples were positive to B. abortus (96.66%).
Inoculation of embryonated eggs. Examination of embryonated eggs revealed the death of chick embryo 4–5 days after inoculation, and the infected yolk sacs were thin-walled, and their blood vessels were severely congested (Fig. 2). Yolk appeared as a bright-colored liquid, and the growth of the embryo was stunted. Embryos were suffering from curled toes, and their bodies were covered with hemorrhage. Only one impression smear of the yolk sac membrane of an egg was found positive for C. abortus. Giemsa stain revealed chlamydial elementary bodies as small stained purple cocci occurring individually and in clusters in the cytoplasm of infected cells.
Identification of C. abortus. In the present study, C. abortus was positive for the ompA gene, based on agarose gel electrophoresis, which demonstrated the expected amplicon of about 1058 bp. The results were affirmed by determining the sequence of the PCR product, and the sequence received the accession number MK643153 in NCBI GenBank and was named C. abortus strain Sul/2017.
Sequence analysis. The sequence of the partial ompA gene of C. abortus strain Sul/2017 showed 99.89% homology with two isolates from the UK with the accession numbers LN554882 and LN554883. However, the isolate in our study showed 99.79% homology with a previous isolate from Sulaimani (Sul/014, accession No. KY399850). Sequence alignment of Sul/2017 with 100 isolates of C. abortus showed that the Iraqi strain had one exclusive single nucleotide polymorphism (SNP, A59050) that caused amino acid substitution A92G in the ompA gene. However, Sul/2017 differed from Sul/2014 by two amino acid substitutions at E115K and K259N.
Phylogenic analysis. The phylogenetic tree topology, based on the ompA gene, showed that Chlamydia Sul/2017 from Iraq belonged to C. abortus (Fig. 3). The phylogenetic tree revealed that Sul/2017 has a common ancestor with isolates from sheep in Iraq and Tunisia with accession numbers KY399850 and HQ62243, respectively. The partial omp1 gene of Sul/2017 has been compared with 75 sequences of Chlamydia genus that were published in GenBank and MLST websites for Chlamydiales (http://pubmlst.org/chlamydiales/). According to the phylogenetic tree, the isolates were distinctly divided into seven clusters; each cluster represented specific species of Chlamydia (Fig. 3). The cluster of C. abortus was subdivided into three groups. The topology of the phylogenetic tree showed that the field isolate Sul/2017 had a common ancestor with Sul/2014, CAAB7, 1H, and Krauss-15 isolates in group 2 of C. abortus.
Abortions by infectious agents in ewes and goats cause considerable economic loss. In addition to Brucella species, Campylobacter fetus subspecies fetus, C. abortus, Salmonella, and Listeria monocytogenes are responsible for abortion in small ruminants (Türütoğlu et al. 2000). In previous surveys, the less-sensitive and -specific complement fixation test had been used, which was described by the World Organization for Animal Health (OIE) as the most commonly used method for serodiagnosis of animals chlamydiosis. However, the technique is tedious, has limited sensitivity, and frequently afflicted by cross-reactions between chlamydial species (McCauley et al. 2007). The recently evolved serodiagnostic tests are primarily based on two main cross-reactive antigens present in all chlamydial species, lipopolysaccharide and the major outer membrane protein (MOMP). Thus, these tests are not species-specific for diagnosing animals infected by OEA. Possibilities for diagnostic detection of chlamydia are better after the introduction of DNA-based methods, particularly the PCR, which permit direct identification from clinical samples and differentiation of species (Longbottom et al. 2001).
In the current study, primers of the C. abortus ompA gene were used; only one sample (3.33%) from aborted ewes was found positive for C. abortus both by yolk sac inoculation and PCR. This result represents the first insight into the presence of C. abortus infection of sheep in Sulaimani province, Iraq, using molecular methods.
Embryonated egg inoculation is considered the gold standard for the isolation of chlamydia. However, the necessity of long-time incubation is its only disadvantage (Condon and Oakey 2007). The findings in this study showed that only one smear from all the impression smear of yolk sac membranes of chicken eggs was found positive for C. abortus by Giemsa stain. Bacterial isolation in embryonated eggs depends on the viable C. abortus elementary bodies in the infected material, which are small and dense. This result was following a study done by Kalender et al. (2013). In this study, the cytological examination of the inoculated egg revealed the typical vascular congestion of yolk sac membranes. The variations in the results of our study and other studies may be attributed to many factors. For example, the geographical location, size and type of samples taken, animal breed, grazing and management strategies, nutritional deficiency, and uncontrolled restriction of diseased animal movement from infected areas are factors that may affect the incidence rate of C. abortus.
The result of this study showed that 29 out of 30 (96.66%) samples were infected with B. abortus using the genus-specific (B4/B5) primers. This result is higher than the outcome of Mohammed et al. (2013), nine out of 15 (60%), and lower than the result of Mukherjee et al. (2007), 19 out of 19 (100%), who detected the Brucella genus using the same primers. This variation may be due to the sampling time from aborted animals (Dağ et al. 2012).
Molecular analysis reveals that C. abortus Sul/2107 strain was the causative agent of abortion in the sheep herd. There was not enough genetic information about C. abortus in Iraq before this study in the GenBank database, except for one isolate (Sul/2014). Genetic analyses indicated that the strain in the present study differed from the previous strain in the region. The number of C. abortus isolates worldwide were 6,718 variable locations documented within the complete phylogeny (Seth-Smith et al. 2017). On the other hand, there were 17,163 variable locations recognized within the phylogeny of C. trachomatis (Harris et al. 2012), and 47,710 variable locations were identified within the strains of C. psittaci (Read et al. 2013). This variation was evident in our study, in which each species of Chlamydia was in a different cluster (Fig. 3). Because of low diversity in C. abortus (Seth-Smith et al. 2017), there were no apparent differences among the strains according to the geographical region or host range within the groups of C. abortus cluster. This outcome made Sul/2017 share a common ancestor with a wide range of strains that originated from different hosts and countries. The grouping within C. abortus was not clear due to a low rate of diversity in the species. Therefore, to make the phylogram more prominent, we used cutoff value with the condensed tree option in the phylogenic tree constriction. Further research is recommended for the whole genome analysis of all Chlamydia strains in Iraq and the neighboring countries due to insufficient genetic information about C. abortus in those countries.
Eman Dhahir Arif https://orcid.org/0000-0002-5591-0410
Phylogenic trees based on the
Phylogenic trees based on the