Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Use of Multiplex PCR for Detection of Campylobacter spp. and their Virulence Factors in Aborted Ewes

Hayder Abdul-Kareem Al-Mutar1* Masar Saab Kadhim2 , Nawaf Nooraldeen Dhaher3, Maythem Abdulealah Ismaeel3

1Department of Surgery and Obstetric, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq; 2Department of Surgery and Obstetric, College of Veterinary Medicine, Al-Qadisiyah University, Iraq; 3Department of Medicine, Surgery and Obstetrics, Veterinary Medicine, Tikrit University, Iraq.

Received | September 07, 2025; Accepted | October 18, 2025; Published | October 28, 2025

*Correspondence | Hayder Abdul-Kareem Al-Mutar, Department of Surgery and Obstetric, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq; Email: [email protected]

Citation | Al-Mutar HAK, Kadhim MS, Dhaher NN, Ismaeel MA (2025). Use of multiplex PCR for detection of Campylobacter spp. and their virulence factors in aborted ewes. J. Anim. Health Prod. 13(s1): 701-705.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.s1.701.705

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Campylobacteriosis is a zoonotic disease, infecting sheep, goat, cattle and human. In human, it causes enteritis, while in sheep and cattle causes reproductive disease (Hlashwayo et al., 2020). Campylobacteriosis in sheep, goats and cattle may cause enteric diseases, which is characterized by watery or bile-streaked diarrhea or may contain blood or mucoid, vomiting and sometimes fever. Other important disease caused by Campylobacter spp. is reproductive disease (Hagos et al., 2021). Campylobacter fetus or Campylobacter jejuni cause abortion in the late stage of pregnancy with gross lesions in the placenta and can cause stillbirths and weak lambs, sometime these cases followed by metritis (Mewari and Chauhan, 2020). Campylobacter sheds in vaginal discharges, aborted fetuses and fetal membranes of aborting sheep (Souhayla et al., 2017; Büyük et al., 2020). Main pathological changes include subcutaneous edema, and in rarely cases blood-stained fluid showed on body cavities, yellow or dark brown exudate and necrotic plaques may be observed on fetal cotyledons, and oedema may occur in the inter-cotyledonary areas (Abdulameer and Saleem, 2018; Rush and Edmondson, 2021). Campylobacter carry many virulence factors, like cytolethal dis­tending toxin B (cdtB), which broadly spared among gram negative bacteria and

 

Table 1: Primers used in the current study for detection of Campylobacter spp.

Gene name	Sequences		Product size	References
Campylobacter fetus	F	5-GGAAGCCGCAGCTGCTAAGAT-3	359	Persson and Olsen, 2005
	R	5-AGCCAGTAACGCATATTATAGTAG-3
Campylobacter Jejuni	F	5-CAAATAAAGTTAGAGGTAGAATGT-3	161
	R	5-CCATAAGCACTAGCTAGCTGA T-3
Campylobacter adherence gene (CadF)	F	TTGAAGGTAATTTAGATATG	400	Yoon, 1999
	R	CTAATACCTAAAGTTGAAAC
Cytolethal-distending-toxin subunit-B gene (CdtB)	F	GTTGGCACTTGGAATTTGCAAGGC	495	Bang et al., 2003
	R	GTTAAAATCCCCTGCTATCAACCA
Cytolethal-distending-Toxin subunit-C (CdtC)	F	CGATGAGTTAAAACAAAAAGATA	182	Rozynek et al., 2005
	R	TTGGCATTATAGAAAATACAGTT
Invasio´n-associated marker 1 (Iam1)	F	GCGCAAAATATTATCACCC	512	Rozynek et al., 2005
	R	TTCACGACTACTATGCGG

 

cause damage and subsequent death on membranes of host cell (Shams et al., 2016). Campylobacter has ability to adhere to host epithelial cell, this process encoded by fibronectin F (cadF) and fibronectin is brokered by CadF fibronectin binding outer membrane protein (Konkel et al., 1997), and the heat survival and stress response proteins htrB and clpP, which are important for survival (Elhadidy et al., 2018).

This study aims to identify and characterize campylobacter species from aborted ewes and to determine the antibiotic resistance markers for the selection of antibiotic use in livestock.

Materials and Methods

A total of 80 samples were collected from aborted ewes (28 from stomach contain of aborted fetus and 52 vaginal swabs collected from aborted ewes in period not more than 48hours after abortion). DNA extraction was using Intron kit on isolate from stomach contain of aborted fetus and vaginal swabs. The PCR amplification was performed using the primers mentioned in Table 1 to detect and confirm Campylobacter spp. and virulence factors.

 

Table 2: Number and ratio of Campylobacter spp detected in current study.

Campylobacter spp.	Number of samples	Number of detection case	Rate of detection
Campylobacter fetus	80	11	13.7%
Campylobacter Jejuni		3	3.7%
Total		14	17.5%

 

Results and Discussion

Out of 80-abortion sample, Campylobacter fetus was detected 13.7% (11/80) of samples while Campylobacter Jejuni was detected in 3.7% (3/80) as in Table 2 and Figure 1.

 

The result of current study differed than result of a previous study (Hamali et al., 2014), where authors have detected of C. fetus subsp. fetus and C. jejuni with a rate of 9% and 1.5% respectively. In another study, (Rizzo et al., 2015) authors have detected of campylobacter spp. with a rate of 3.5%. This difference might be due to difference in the geographical region, number of samples, clinical signs, age and species of animals. The detection of Campylobacter spp. in abortion case endorses that campylobacter spp. is one of important etiology of abortion in sheep. Many studies (Mannering et al., 2006; Chon et al., 2020; Dorsch et al., 2021; Şevik, 2021), detected Campylobacter spp. from abortion ewes. C. fetus and of C. jejuni caused infertility, early embryonic death and a prolonged lambing season. C. fetus caused abortion in the lasted pregnancy period, stillbirths and weak lambs. In some cases, metritis and occasionally deaths may occur (Dawson et al., 2020). Our findings showed that the detection rate of C. fetus more than detection rate of C. jejun, which agreed with result of another study (Tamura and Kumar, 2004). The application of multiplex PCR for

 

Table 3: Number and Rate of Campylobacter spp isolates possessing virulence factor.

Campylobacter virulence factors	Total isolate	DNA out product size	Number of isolates possessing virulence factor	Rate of isolates possessing virulence factor
Campylobacter adherence gene (cadF)	14	400	14	100%
Cytolethal distending toxin subunit B gene (cdtB)	14	495	11	78.5%
Cytolethal distending Toxin subunit c (cdtC)	14	182	12	85.7%
Invasio´n-associated marker 1 (Iam1)	14	512	1	7.1%

 

the detection of campylobacter virulence revealed that cadF, cdtB and cdtC were detected with a rate of 100%, 78.5% and 85.7% respectively while Iam1 detection rate was 7.1% as in Table 3 and Figure 2.

 

 

 

The UPGMA method was used to infer evolutionary history, the optimum A tree having a total of branches lengths of 0.06369197 was depicted. The tree was displayed in scale, with lengths of branches (below the branches) in the same units as the evolutionary distances employed. To reconstruct the genomic tree, the evolutionary distances were calculated using the maximal composite likelihood approach (Tamura et al., 2013) and were expressed in terms of base substitutions made at each site. There were 13 nucleotide sequences in the analysis. The codon locations listed were first, second, third, non-coding. Every position with gaps or the missed information was deleted. The final dataset contained a total of 560 locations. MEGA6.0 was used to carry out evolutionary analyses (Igwaran and Okoh, 2020) Figure 3. The appearance of this virulence factors agreed with study conducted previously (Lluque et al., 2017; Ghunaim et al., 2015). The cadF is one of important genes, which is responsible for heat survival, and translate a protein necessary for adhesion and invasion (García-Sánchez et al., 2018). The cdtB genes is tripartite and fully functional toxin and plays function in causing harm to cells that caused dame in mucosa, Additionally, it has DNaseI-like activity which caused break down of double strand DNA (Al-Mutar, 2017). the cdtC gene is cytotoxin, causes cell distraction by DNA damage, chromatin and fragmentation (Ghorbanalizadgan et al., 2018). In the current study C. jejuni was isolated in low rate, proportionate to Iam1 gene which was also detected in the lowest rate This supports the scientific references that refer to that Iam1 is frequency in C. jejuni (Rozynek et al., 2005).

Conclusion

Campylobacteriosis is the leading cause of ewe abortion in the area. C. fetal is the most pathogenic Campylobacter species in the studied region, affecting 17.5% of ewes.

ACKNOWLEDGEMENT

The author appreciates the laboratory facilities offered by the College of Veterinary Medicine, University of Baghdad; Al-Qadisiyah and Tikrit.

NOVELTY STATEMENT

This study introduces an innovative approach to clinical besides genetic analysis of bacteria in aborted ewes, and determines the antibiotic resistance.

AUTHOR’S CONTRIBUTION

This study was designed and written by the authors.

Funding

None.

Data availability

The publication includes all datasets created and analyzed during the investigation.

Ethics statement

Not applicable.

Generative AI and AI-assisted technology statement

The authors acknowledge that the primary purpose of generative AI tools (such as ChatGPT and software language editing) is to improve formatting, grammar, and language clarity. No artificial intelligence (AI) techniques were employed for data interpretation, analysis, or scientific conclusion-making. The content of this article is entirely the writer’s responsibility.

Conflict of interest

The author have declared no conflict of interest.

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