Prevalence and Identification of Infectious Abortion Pathogens in Sheep Flocks of North Khorasan, Iran

1. Introduction

The small ruminant industry plays a significant role in livestock production, particularly in the production of sheep meat, which is the most popular meat consumed by the Iranian population ( 1 ). The primary objective of ovine rearing in Iran is the production of meat, with an annual output of 265,000 tons ( 2 ). The potential profitability of meat production in this country is significant. Nonetheless, the species may face potential threats from various factors, including reproductive failure, which has the capacity to significantly diminish both meat and milk production. Abortion has been identified as a significant contributing factor to reproductive setbacks in flocks of small ruminants, encompassing both infectious and non-infectious underlying causes. An array of agents, comprising bacteria, viruses, fungi, and protozoa, have been identified as potential contributors to abortion. Furthermore, non-infectious etiologies such as trauma, elevated body temperature, concentrated densities, toxicities from plant origins, and nutrient deficiencies are prevalent ( 3 ). Brucella melitensis is the principal infectious agent responsible for economically significant cases of abortion in Iran and various other countries ( 4 ). Furthermore, global instances of abortion caused by other pathogens, including Toxoplasma gondii, Campylobacter spp., Chlamydia abortus, and Coxiella burnetii, are frequent ( 5 ). It is noteworthy that viral agents such as the Blue Tongue Virus, Border Disease Virus, and Akabane virus are prevalent in regions where small ruminants are commonly used as a source of meat ( 5 ). In order to consider preventive measures and reduce economic losses, it is necessary for each country to estimate the presence of abortifacient agents in different geographical areas. In the domain of discerning and identifying the primary culprits behind the occurrence of miscarriages, the utilization of precise techniques boasting impeccable sensitivity and specificity assumes paramount significance. The present study sought to utilize ELISA, conventional PCR, and bacteriological examination to assess the presence of the most common abortifacient pathogens among sheep flocks in North Khorasan, Iran.

2. Materials and Methods

2.1. The structural Configuration of the Research Investigation

From 2016 to 2017, an investigation was conducted to ascertain the etiology of fetal losses in ovine flocks located in North Khorasan. The subjects of the study were sheep of the Moghani breed, and the cases examined were devoid of any placenta. A comprehensive examination of a total of 133 fetuses was undertaken, employing bacteriological culture, ELISA, and PCR methods in pursuit of identifying the precise causes of abortion. The examination protocol encompassed the evaluation of all 133 fetuses for the presence of BDV, BTV, and E. coli. Additionally, an assessment of other potential pathogens was conducted on 117 fetuses out of the original 133. The laboratory received the aborted fetuses while adhering strictly to the principles of cold chain logistics, ensuring proper preservation utilizing adequate ice pack distribution.

2.2. Sample Collection

The fetuses were necropsied in a sterile manner, and then, fetal fluids were extracted from the thoracic and pericardial cavities using sterile syringes for the ELISA test. A variety of parenchymatous tissues and fluids, including those from the liver, spleen, kidneys, lung, and abomasal contents, were obtained with the objective of subjecting them to microbiological culture and polymerase chain reaction (PCR) analysis.

2.3. ELISA

Sera from aborted fetuses were subjected to testing for antibodies against C. burnetii, C. abortus, and T. gondii using a commercial indirect ELISA kit from IDvet, France. Infections with BDV and BTV were examined using a competitive ELISA kit from the same manufacturer, following their instructions.

2.4. PCR Assay

A CinnaGen DNA extraction kit was utilized to extract DNA from both the abomasal contents and the homogenized tissues. The detection of B. melitensis, Salmonella, Campylobacter spp., C. abortus, C. burnetii, T. gondii, L. monocytogenes, and Leptospira spp. was conducted through conventional PCR using methods that had been previously established and genus- and species-specific primers that had been cited in the literature.

2.5. Bacteriological Procedure

Consequently, bacteriological procedures were performed for the detected agents, based on the positive results of the PCR. However, the procedure of isolation was generally conducted for the purpose of diagnosing every involved pathogen. Subsequent to initial plating onto blood agar and MacConkey agar (Merck, Germany), the homogenized tissues were incubated aerobically and anaerobically for 48 hours at 37°C in pairs of plates. Following a 24-hour incubation period, an inspection of the bacterial growth was conducted, and colonies of interest were subsequently subjected to a series of biochemical tests. These tests were based on colony morphology and gram staining results, with the objective of obtaining and identifying pure bacterial cultures.

3. Results

The utilization of diverse diagnostic methodologies in this study yielded the identification of various pathogens, including C. abortus, E. coli, B. melitensis, Salmonella, Campylobacter, Leptospira, C. burnetii, T. gondii, BDV, and BTV, in the aborted fetuses (see Table 1 for details). T. gondii (12%), BTV (9%), BDV (3%), and C. burnetii (2.5%) were identified in fetal serology. The following were detected by PCR: C. abortus (25.6%), C. burnetii (14.5%), B. melitensis (12%), Salmonella spp. (8.5%), T. gondii (6.8%), Leptospira spp. (2.5%), and Campylobacter spp. (1.7%). Furthermore, positive results from the polymerase chain reaction (PCR) for B. melitensis, Salmonella spp., Campylobacter spp., Leptospira spp., L. monocytogenes, and E. coli were utilized for bacteriological examination. B. melitensis (12%) and E. coli (9%) were isolated using bacteriological culture media. C. abortus was identified as the most prevalent agent in 25.6% of the studied fetuses. It was observed that all PCR-positive results were not consistent with the ELISA method and bacteriological procedures (Table 1). Furthermore, Brucella isolates in culture from fetuses were also confirmed by PCR, with all 15 samples yielding positive results.

4. Discussion

The present study identified some of the most prevalent abortifacient pathogens in sheep and identified C. abortus as the primary agent, accounting for 25.6% of cases (Table 1). C. abortus has been identified as the primary cause of abortion in various countries and poses a significant global challenge to the reproduction of small ruminants. This gram-negative bacterium has been identified as the causative agent of economic losses across Europe, North America, and Africa, accounting for 45% of ovine abortions in the UK ( 14 ) In their 2022 study, Esmaeili et al. observed bacterial involvement in 46.9% of small ruminant abortion cases in Iran, with C. abortus, B. melitensis, and C. burnetii notably implicated, particularly in instances where abortion rates exceeded 10% ( 15 ). The present data, when considered in conjunction with the preceding report, indicate that enzootic ovine abortion is an endemic disease in our nation ( 15 ). Given the direct correlation between infection rates and flock hygiene, educational initiatives should be implemented to mitigate the incidence of new infections, particularly in rural areas where rearing systems are less sophisticated. Fetal serology has been demonstrated to be a valuable diagnostic tool for the identification of Chlamydia-specific antibodies. It is evident that maternal antibodies are incapable of reaching the fetus through the placenta, while fetal antibodies are derived from uterine exposure. Consequently, the detection of any antibody titer is presumed to indicate the presence of the abortifacient chlamydial species, such as C. abortus. A comparable study conducted in Northern Ireland in 2001 revealed the presence of anti-Chlamydia antibodies in 70 out of 417 fetal blood and thoracic fluid samples ( 16 ). In the present study, while PCR revealed a high rate of fetal infection with C. abortus (Table 1), no antibody reaction was detected in the samples. This finding underscores the necessity of employing more sensitive methodologies with suitable samples. The Iran Veterinary Organization has initiated a national plan to address brucellosis, recognizing its significant role in small ruminant abortions in the country. As part of this plan, the reduced-dose Rev-1 vaccine against B. melitensis has recently been excluded, while concerns persist about the limited duration of immunity provided by the full-dose Rev-1 vaccine compared to the reproductive lifespan of ewes. Despite the implementation of vaccination programs, B. melitensis continues to pose a significant challenge to small ruminant flocks. Current data demonstrate that the bacterium is detected in 12% of aborted fetuses, with both polymerase chain reaction (PCR) and isolation methods employed in the diagnostic process (Table 1). In the present study, a polymerase chain reaction (PCR) analysis was conducted, which revealed C. burnetii as the second most frequently detected bacterium (14.5%). Mostafavi et al.'s 2019 study emphasizes the public health significance of Q fever, caused by C. burnetii, in different regions of Iran, often resulting from direct or indirect contact with ruminants ( 18 ). In the absence of vaccination, the implementation of strict biosecurity measures is imperative for the control of coxiellosis ( 19 ). As indicated by the findings presented in Table 1, the polymerase chain reaction (PCR) method demonstrated heightened sensitivity in comparison to the enzyme-linked immunosorbent assay (ELISA). Furthermore, the presence of the C. burnetii antibody was detected in a mere 2.5% of the fetal samples examined. The utilization of serological methodologies may result in the underestimation of infected cases, given the constrained validity of these approaches in detecting C. burnetii and the potential variability in the sensitivity of commercially available ELISA kits. Salmonella spp. was identified through the polymerase chain reaction (PCR) in 8.5% of the samples examined in this study. Given its association with abortion and lamb mortality, it is imperative to incorporate preventive measures and carrier identification into the abortion control plan ( 20 ). We then proceeded to examine the PCR-positive samples by bacteriological procedures (Table 1). The negative result obtained from the isolation method indicated that PCR is a more sensitive method, despite the fact that isolation has high specificity and is generally considered a gold standard. Consequently, negative results from bacteriological samples must be confirmed by molecular methods. The association between infection with Leptospira spp. and reproductive failure in small ruminants, particularly goats, is well-documented ( 21 ). In the present study, polymerase chain reaction (PCR) analysis detected the bacterium in 2.5% of the samples, while Campylobacter spp. was not isolated through bacteriological procedures. However, DNA from the bacterium was detected in only 1.7% of the fetuses, indicating a lower rate of fetal infection with the bacterium. A similar phenomenon was observed in an abortion outbreak in Iran in 2022, where Campylobacter spp. was isolated in 3.3% of fetuses. This occurrence was less frequent when compared to other bacterial abortifacients, including C. abortus, C. burnetii, and B. melitensis ( 22 ). In the present study, E. coli was isolated from 9% of fetal samples. However, the bacterium is often neglected in abortion cases. The seroprevalence of T. gondii infection was found to be 12%. However, DNA from T. gondii was identified in 6.8% of the fetuses. As the parasite is lethal to ovine fetuses and has zoonotic potential, determining the infected animals is crucial. Currently, there is no approved vaccine available in Iran. In such circumstances, the implementation of strategies is imperative to prevent exposure of small ruminants to stray cats and to mitigate the likelihood of cats accessing the animals' feed ( 23 ). Border disease (BD) is recognized as endemic among small ruminants in Iran; however, there is a paucity of studies focused on the detection of BDV in sheep and goats. The results of the study indicated that 3% of the fetuses were infected with BDV. Given ELISA's sensitivity and value for virus detection in fetal fluid ( 24 , 25 ), the present study emphasizes the assessment of seroprevalence in other regions of Iran. Furthermore, an analysis was conducted to ascertain the prevalence of BTV among the flocks, and the virus was detected in 9% of the fetuses. A previous study conducted by Esmaeili et al. in 2022 reported BTV in 1.9% of aborted fetuses in Iran ( 15 ). The present study employed a combination of PCR, ELISA, and bacterial isolation methodologies to investigate the etiology of abortion in fetal tissues and fluid. However, the identification of L. monocytogenes was not successful; consequently, it is necessary to utilize a variety of samples, including placenta from additional aborted animals. Given the zoonotic nature of many abortion causes of small ruminants; vaccination programs must be implemented as a primary strategy. Furthermore, given that the majority of sheep in Iran are managed under extensive farming systems, the absence of adequate educational resources among farmers can compromise the efficacy of any abortion control initiative. The present study demonstrated the necessity for further investigation, which can be accomplished through the utilization of straightforward and sensitive methodologies such as molecular tests. Moreover, additional epidemiological and risk factor research is necessary to further elucidate the etiology of ovine abortion.

Acknowledgment

The authors express their sincere gratitude and appreciation to all the ranchers and personnel of the North Khorasan Veterinary Head Office, Bojnurd, who contributed to this study.

Authors' Contribution

Study concept and design: H. E and H.GH

Acquisition of data: H. E, P.B and M.SH

Analysis and interpretation of data: H. E.M.SH and M.H

Drafting of the manuscript: H.E and M.H

Administrative, technical, and material support: P.B and M.SH

Study supervision: H. E and H.GH

Ethics

The authors of this study affirm that all ethical standards were upheld in the preparation of the submitted article.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

The present study was conducted with financial support from the Iran Veterinary Organization.

Data Availability

The data produced and/or analyzed during the present study can be obtained from the corresponding author upon request.

References

1. Esmaeili H, Ghorani M, Baghal Arani E, Shakeri AP. Detection of contagious ovine ecthyma (orf) and risk factors for infection in small ruminants in Iran. Comparative Immunology, Microbiology and Infectious Diseases. 2021; 79: 101714.
2. Agriculture Ministry. Diseases of sheep. California: John Wiley & Sons; 2020.
3. Noakes DE, Parkinson TJ, England GC. Arthur's Veterinary Reproduction and Obstetrics. Amsterdam: Elsevier publishing; 2016.
4. Esmaeili H, Tajik P, Ekhtiyarzadeh H, Bolourchi M, Hamedi M, Khalaj M, Amiri K. Control and eradication program for bovine brucellosis in Iran: an epidemiological survey. Journal of Veterinary Research. 2012; 67: 211-221.
5. Van Engelen E, Luttikholt S, Peperkamp K, Vellema P, Van Den Brom R. Small ruminant abortions in the Netherlands during lambing season 2012-2013. Veterinary Record. 2014; 174: 506.
6. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. Journal of Clinical Microbiology. 1994; 32: 2660-2666.
7. Rahn K, De Grandis S, Clarke R, McEwen S, Galan J, Ginocchio C, Curtiss Iii R, Gyles C. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes. 1992; 6: 271-279.
8. Bang DD, Pedersen K, Madsen K. Development of a PCR assay suitable for Campylobacter Spp. mas screening programs in broiler production. Journal of rapid methods and automation in microbiology. 2001; 9: 97- 113.
9. Laroucau K, Souriau A, Rodolakis A. Improved sensitivity of PCR for Chlamydophila using pmp genes. Veterinary microbiology. 2001; 82: 155- 164.
10. Hoover T, Vodkin M, Williams J. A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence. Journal of Bacteriology. 1992; 174: 5540-5548.
11. Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. Journal of Clinical Microbiology. 1989; 27: 1787-1792.
12. Gouws PA, Liedemann I. Evaluation of diagnostic PCR for the detection of Listeria monocytogenes in food products. Food Technology and Biotechnology. 2005; 43: 201-205.
13. Stoddard RA, Gee JE, Wilkins PP, McCaustland K, Hoffmaster AR. Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene. Diagnostic Microbiology and Infectious Disease. 2009; 64(3): 247-55.
14. Longbottom D, Coulter L. Animal chlamydiosis and zoonotic implications. Journal of Comparative Pathology. 2003; 128 (4): 217-44.
15. Esmaeili H, Shakeri A, nosrati Rad Z, Baghal Arani E, Villanueva-Saz S, Ruiz H, Lacasta D. Causes of abortion in Iranian sheep flocks and associated risk factors. Veterinary Research Communications. 2022; 46: 1227-1238.
16. Kennedy HE, McCullough SJ, Graham D, Cassidy G, Malone FE, Ellis WA. Detection of Chlamydial antibody by fetal serology- an aid to the diagnosis of ovine abortion. Journal of Veterinary Diagnostic Investigation. 2001; 13: 30-35.
17. Ghorani M, Esmaeili H. Comparison of susceptibility of different goat breeds to live attenuated goatpox vaccine. Small Ruminant Research. 2022; 212: 106721.
18. Mostafavi E, Molaeipoor L, Esmaeili S, Ghasemi A, Kamalizad M, Yousefi Behzadi M, Naserifar R, Rohani M, Hashemi Shahraki A. Seroprevalence of Q fever among high-risk occupations in the Ilam province, the west of Iran. PLoS One. 2019; 14 (2): 1-10.
19. Plummer PJ, McClure JT, Menzies P, Morley PS, Van den Brom R, Van Metre DC. Management of Coxiella burnetii infection in livestock populations and the associated zoonotic risk: a consensus statement. Journal of veterinary Internal Medicine. 2018; 32 (5): 1481-1494.
20. Esmaeili H, Kalateh Rahmani H. Detection of Salmonella carriers in sheep and goat flocks of Bushehr and Lorestan provinces, Iran. Journal of Medical Bacteriology. 2016; 5: 50-53.
21. Elvira Partida L, Fernández M, Gutiérrez J, Esnal A, Benavides J, Pérez V, Torre A, Alvarez M, Esperon F. Detection of bovine viral diarrhoea virus 2 as the cause of abortion outbreaks on commercial sheep flocks. Transboundary and Emerging Diseases. 2017; 64:19-26.
22. Hazlett MJ, McDowall R, DeLay J, Stalker M, McEwen B, van Dreumel T, Spinato M, Binnington B, Slavic D, Carman S. A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens. Journal of Veterinary Diagnostic Investigation. 2013; 25: 359-368.
23. Gangneux FR, Darde ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clinical Microbiology reviews. 2012; 25 (2): 264-296.
24. García-Pérez AL, Minguijon E, Estevez L, Barandika JF, Aduriz G, juste RA, Hurtada A. Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype). Research in Veterinary Science. 2009;  86: 345-352.
25. Rasooli A, Nouri M, Seyfi Abad Shapouri MR, Mohseni-Parsa S, Baghbanian HR, Lotfi M, Daghari M. Serological Detection of SRMV, BVDV, BHV-1and BEFV in Camels (Camelus dromedarius) in Southwest Iran. Iranian Journal of Veterinary Medicine. 2023; 17 (2): 139-148.