Detection of Campylobacter fetus in aborted ewes in Sulaimani province by PCR

Highlights:

1. The research highlights through detection of the Campylobacter fetus from sheep flocks in the Sulaimani Province.

1. PCR assay had used in this project, which is considered as more specific and accurate.
2. Campylobacter fetus was isolated from aborted fetuses and vaginal swab samples.
3. In Sulaimani Province, it has been concluded that the Campylobacter fetus is a significant cause of abortion of ewes.

Detection of Campylobacter fetus in aborted ewes in Sulaimani province by PCR

E. D. Arif

Department of Microbiology, College of Veterinary Medicine, University of Sulaimani, Sulaymaniyah, Iraq

[email protected], 0000-0002-5591-0410

Abstract

Abortion is one of the most critical factors affecting lambing rates and, as a result, sheep farm profitability. It is also significant from a zoonotic viewpoint, in addition to financial losses. In sheep flocks, Campylobacter fetus causes infectious infertility, embryonic death, and miscarriages. The study investigated C. fetus from aborted fetuses and vaginal swab samples collected from sheep flocks in the Sulaimani province by the polymerase chain reaction. Thirty-eight aborted fetuses and 70 vaginal swabs were collected from sheep flocks in three districts of Sulaimani province (Kalar, Said Sadiq, and Chamchamal) from March 2018 to June 2019. The pathogen was identified in clinical specimens using conventional PCR. C. fetus was isolated in 16 of 38 aborted fetuses (42.1%) and 13 of 70 vaginal swabs from aborted ewes (18.6 %). The C. fetus gene 16S rRNAwas sequenced and received the accession number MW694741 in NCBI GenBank. Phylogenetic analysis of 16S rRNA gene sequences designated that the C. fetus isolates formed a separate branch displayed the highest similarity and clustered with MN203686.1 and EU773268.1 accessions in a specific clade. A lower degree of affinity of C. fetus was revealed with Campylobacter coli and Campylobacter jejuni.

Keywords:Campylobacter fetus, Abortion, Sheep, PCR

الکشف عن العطیفة الجنینیة فی النعاج المجهضة فی محافظة السلیمانیة بوساطة تفاعل البلمرة المتسلسل

ایمان ظاهر عارف

فرع الاحیاء المجهریة، کلیة الطب البیطری، جامعة السلیمانیة، السلیمانیة، العراق

الخلاصة

یعتبر الإجهاض من أهم العوامل التی تؤثر على معدلات الحمل، وناتج ذلک على ربحیة حقول الأغنام. کما أنها مهمة من وجهة نظر انها ذات منشأ حیوانی. بالإضافة إلى الخسائر المالیة. فی قطعان الماشیة والأغنام، تسبب العطیفة الجنینیة عقم معدی، موت جنینی وإجهاضات. تم التحری عن العطیفة الجنینیة فی أجنة النعاج المجهضة وعینات المسح المهبلیة التی تم جمعها من قطعان الأغنام فی محافظة السلیمانیة بوساطة تفاعل البلمرة المتسلسل. جمع 38 جنینا مجهضا و 70 مسحة مهبلیة من النعاج فی ثلاث اقضیة من محافظة السلیمانیة (کلار، سید صادق وجمجمال) من اذار 2018 إلى حزیران 2019. استخدم تفاعل البلمرة المتسلسل لتحدید المسبب المرضی. تم عزل العطیفة الجنینیة فی 16 من 38 من الأجنة المجهضة بنسبة 42.1٪ و 13 من 70 مسحة مهبلیة من النعاج المجهضة بنسبة 18.6٪. تم تحدید التسلسل الجینی للحامض النووی الرایبوزی نوع S16للعطیفة الجنینیة وحصل على رقم الانضمام MW694741 فی بنک جینات ان سی بی ای. حدد التحلیل الوراثی لتسلسل الجینی لحامض النووی الرایبوزی نوع S16 إلى أن العطیفة الجنینیة المعزولة شکلت فرع منفصل وعرض أعلى تشابه وتم تجمیعها مع مدخلات MN203686.1, EU773268.1 فی شفرة محددة. کانت اقل درجة تقارب لجرثومة العطیفة الجنینیة مع العطیفة القولونیة والعطیفة الصائمیة.

Introduction

Abortion is one of the most significant matters in sheep breeding, and it is financially damaging to the farmer (1,2). In economies where lamb is the primary source of animal protein, ovine abortion is essential (3,4). Different bacterial, viral, and protozoal diseases have been associated with infectious ovine abortions. These infections are critical in terms of public health and the economy. Brucellosis, campylobacteriosis, chlamydiosis, and salmonellosis are the most common bacterial infections that cause abortion (5,6). Campylobacter was secluded from aborted sheep fetuses in 1909, and the name Campylobacter was given to it in 1963. These organisms cause abortion in cows and ewes, as well as severe enterocolitis in humans. C. fetus subsp. fetus or Campylobacter jejun are the source of this disease. Both organisms are capable of causing abortion epidemics indicated by substantial lesions in the placenta, fetal tissues, or both (7-9). Campylobacter jejuni, C. coli, and C. fetus subsp. fetus is common in the world and causes reproductive diseases in sheep. They are Gram-negative, motile and microaerophilic. Environmental samples such as soil, water, and food can be contaminated with Campylobacter species resulting from contact with contaminants such as feces and aborted fetuses (10,11). Abortion, stillbirths, and the birth of weak lambs are some of the clinical signs of campylobacteriosis in sheep (12-14). This disease spreads in flocks by introducing new carrier animals, and pregnant ewes could become infected by drinking contaminated water or ingesting contaminated feed (15-17). Campylobacter fetus is now considered a zoonotic disease. Human infections are considered to be introduced through cattle and sheep products. Human infection with C. fetus usually begins with the bacteria being consumed orally, followed by colonization of the intestine. Some colonized individuals induce diarrhea. Occasionally, C. fetus causes severe systemic infections (18-20). Rapid diagnosis of an agent causing abortion is of great importance in preventing and controlling the disease. Old diagnostic methods of campylobacteriosis are time-consuming, somewhat challenging, and are not always accurate. Thus, molecular methods like PCR have been welcomed in recent years, particularly in research studies (21,22). This study aimed to investigate C. fetus, one of the critical abortion agents, from aborted sheep fetuses and vaginal swab samples collected from sheep flocks in the Sulaimani province by PCR.

Materials and methods

Ethical approval

The study was carried out in the Research Center, College of Veterinary Medicine, University of Sulaimani, from March 2018 - June 2019 and approved by the Ethical Committee (Approval No. 01268/20Feb2018).

Study area and sample collection

Between March 2018 and June 2019, 108 samples were obtained from various flocks in three districts of Sulaimani province with a history of abortion. We collected 38 samples of aborted fetuses. Eighteen samples had been from Kalar, 13 from Said Sadiq, and seven from Chamchamal. In addition, 70 vaginal swab samples had been taken from the vaginas of aborted ewes, 38 from Kalar, 12 from Said Sadiq, and 20 from Chamchamal. In these districts, the sheep flocks management method is traditional; different people own the animal flocks. Indoors, the sheep are fed grain, hay, and silage before being released to graze on pasture. Various animal species might graze on the same pasture, or flocks might share rams to enhance fertility. Tissue from recently aborted fetuses (liver and spleen) and their dams (vaginal swabs) with abortions within the previous 2–4 days was obtained using disposable blades and scissors. Collected samples were placed in plastic containers, labeled, and sent in a refrigerated box to the Research Center of the College of Veterinary Medicine / University of Sulaimani, where they were identified as C. fetus the same day.

Extraction of DNA

A DNA extraction kit was used on the samples to extract the DNA (GeNet Bio, South Korea). Following the manufacturer's instructions, the process was carried out. DNA quality was measured spectrophotometrically, and low concentration samples (lower than 100 ng/μL) were eliminated from further analyses (23).

Oligonucleotides and PCR amplification

Hossein et al. (24) presented the primers used for amplifying a 265 bp segment of the 16S rRNA gene, with forward (5´-TTTGTTAGGGAAGAACCATG-3') and reverse (5´-CGCAATGGGTATTCCTGGT-3') primers. Macrogen® (South Korea) progressed the primers for our research. The total DNA was amplified using PCR Add Start Taq Master (PCR Add Start Taq Master) (Korea, Add bio). 0.2 mL PCR tubes were used for the experiment. The PCR tube included l0 µL of master mix, 5 µL of DNA, and l µL (10 pmol) of each forward and reversed primer. The ultimate volume of 20 µL was achieved by adding 3 µL of DEPC-treated water (25, 26). The thermal cycler method began with an initial denaturation at 95 °C for five minutes. The samples were then subjected to 30 cycles of denaturation (95°C for 1 minute), annealing (55°C for 1 minute), and extension (72°C for 1 minute), a five-minute final extension at 72°C was also included. The PCR products were examined after loading 7 µL on a 1 % agarose gel in 1× Tris/Borate/EDTA (TBE) buffer (27,28). A 5 µL safe dye was used to stain the gel. Using the Safe-Blue Illuminator/Electrophoresis System, electrophoresis was performed for 50 minutes at 120 volts. By comparing PCR result amplicons to a 100 bp DNA ladder, migration patterns were studied (29,30).

Sequencing of the 16S rRNAgene and phylogenetic analysis

South Korea's Macrogen Sequencing Facility sequenced the PCR results of the C. fetus16S rRNA gene. After being sequenced many times, the gene sequences were submitted to GenBank to authenticate each nucleotide's identification to get accession numbers. Prokaryotes were identified and classified using a sequencing study of the small subunit ribosomal RNA (16S rRNA)gene. Microbial diversity is also estimated using the sequence of 16S rRNA genes amplified from the environment. The pathogen causing intestinal disease and abortions in sheep, C. fetus, was isolated from northern Iraq in 2018. The partial 16S rRNA gene sequence (265 bp) of the strain was collected from the vaginal swabs of the aborted ewes, and the two strands were sequenced. The sequence was compared with those in available databases using BLAST and aligned with its nearest neighbors using Mega X (31-33).

Results

Samples

Genomic DNA was successfully extracted from fetal samples and vaginal swabs of aborted ewes. In the current investigation, 108 samples were taken from Kalar, Said Sadiq, and Chamchamal, where abortions had been observed. Sixteen samples (42.1%) from aborted fetuses and 13 samples (18.6 %) from vaginal swabs of PCR were used to identify C. fetuses in aborted ewes (Table 1).

Table 1: PCR results for the detection of Campylobacter fetus in aborted fetuses and vaginal swabs of aborted ewes

Identification of Campylobacter fetus

According to agarose gel electrophoresis, the campylobacter fetus was positive for the 16S rRNA gene in the present study, which indicated a 265 bp amplicon (Figure 1). The sequencing of the PCR product was determined to corroborate the results, which was given the NCBI GenBank accession number MW694741 and given the name C. fetus.

Figure 1. Specific amplification of target DNA from Campylobacter by PCR using specific primers. Lane M: Show 100 bp DNA size marker, lane 1: Negative control (no DNA in the PCR reaction mix), lane 2-9: An aborted ewe's samples (265bp).

Phylogenetic analysis

The taxonomic position of C. fetusas revealed by neighbor-joining analysis of the 16S rRNA gene sequence alignment (Figure 2). All accessions were clustered into three main classes. 16S rRNA gene sequence phylogenetic analysis showed that the C. fetus isolates formed a separate branch, displayed the highest similarity, and clustered with uncultured Campylobacter species (Accession number MN203686.1) uncultured bacterium BHSD (Accession number EU773268.1) in a specific clade. C. fetus has a reduced affinity for the Campylobacter coli and Campylobacter jejuni bacteria.

Figure 2. A maximum-likelihood dendrogram based on 16S rRNA gene sequences revealed the phylogenetic relations between C. fetus and some similar taxa. In branch nodes, bootstrap values based on 100 replicates are presented. Bar = 0.005 substitutions per nucleotide position.

Discussion

Abortions caused by infectious agents in sheep breeding are a fundamental problem. These agents lead to significant economic losses, a loss of offspring, a decrease in milk yield, a decrease in breeding value, and infertility (34). In many places, campylobacteriosis is a significant cause of sheep abortion. The majority of abortions occur in the final month of pregnancy. Infection causes include the placenta, the fetus, the birth fluids, the vaginal discharge, and the feces of the ewe (35). In the present study, vaginal swabs from aborted ewes and aborted fetuses were investigated by PCR to detect Campylobacter. Many studies previously used this method (24,36).

According to our data, in sheep, campylobacteriosis causes abortion. Moreover, C. fetus is the most common cause of ewe abortion in Sulaimani province. C. fetus was isolated from42.1% of aborted fetusesand 18.6% of vaginal swabs from aborted ewes using PCR. Compared to traditional bacteriological tools, rapid identification of the causal agent utilizing molecular techniques shows the effectiveness of PCR techniques as a practical alternative to laboratory diagnosis. This is especially significant when it comes to detecting fastidious microorganisms like the Campylobacter genus (37). Detection rates in the present study are considerably higher than published research on abortion in sheep with infection of C. fetus from other countries such as Iran 7.7% (24) and Turkey 7% (38). Differences between studies may be attributed to geographical region, diagnostic techniques, the animal's breed, collection, and timing of materials. On the other hand, our research results contradict those of other studies (7,39), which detected that C. jejuni and C. coli were the most prevalent campylobacter species in sheep diseases and abortion in the United States and Turkey, respectively.

The current study recorded C. fetus isolated from aborted ewes for the first time in Iraq, and it was registered in the GenBank database (Ac.: MW694741). Focused on the 16S rRNA gene, all accessions were clustered into three main classes: the C. fetus isolates. The present study formed a separate branch and showed the highest similarity with uncultured Campylobacter spp. (Accession number MN203686.1) and uncultured bacterium BHSD (Accession number EU773268.1). Both MN203686.1 and EU773268.1 were isolated from the same animal (sheep) but in different countries. According to Mohakud et al. (40) and Ley et al. (41), the closely related isolates MN203686.1 and EU773268.1 were isolated in the feces of sheep. Regarding these findings, the present study suggests further future studies to detect a genetic relation between C. fetuses isolated from the vagina andthose isolated from sheep feces. Because of limited genetic information on C. fetus in those states, future studies should focus on whole-genome sequencing of all Campylobacter strains in Iraq and surrounding countries.

Conclusion

Campylobacteriosis is a significant cause of abortion of ewes in Sulaimani province, accounting for the majority of sheep abortions in the area. C. fetus is the most pathogenic Campylobacter species in our region, causing 42.1 % of ewe abortions.

Acknowledgments

The author would like to thank the College of Veterinary Medicine, University of Sulaimani, Sulaymaniyah, Iraq.

Conflict of interest

The author declares that he has no conflict of interest.