جداسازی و شناسایی باکتری بروسلا با استفاده از روش PCR در سگ‌های بومی شهرستان نیشابور و حومه

1. Introduction
Brucellosis is a globally distributed zoonotic disease that continues to impose significant economic and public health burdens in many developing countries, including Iran. The disease is caused by facultative intracellular bacteria of the genus Brucella, with Brucella melitensis considered the most virulent species affecting humans and small ruminants such as sheep and goats [1, 2]. Infected animals typically shed the organism through reproductive discharges, including placental tissues, aborted fetuses, and vaginal secretions, contaminating the environment and facilitating transmission through direct contact or ingestion [3, 4]. While cattle, sheep, and goats are recognized as the primary reservoirs of brucellosis, dogs, particularly those used in herding or guarding livestock, can also become infected. These animals are frequently exposed to contaminated tissues during parturition or abortion events in infected herds. As a result, they may act as asymptomatic carriers of B. melitensis, posing a zoonotic risk to humans, especially those living or working in close contact with farm animals [5, 6]. Evidence from recent Iranian studies confirms the presence of B. melitensis biovars in dogs, emphasizing their potential role in the complex epidemiology of brucellosis [5].
In Iran, brucellosis remains endemic in many regions, including the northeast. The province of Khorasan Razavi, with its extensive livestock farming and transboundary animal movements, is considered a high-risk zone [5, 6]. Despite the national vaccination program targeting livestock, sporadic outbreaks continue to occur, and the role of secondary reservoirs, such as dogs, is often overlooked in surveillance efforts. Traditional serological tests, such as the Rose Bengal plate test (RBPT), are commonly used for initial screening due to their simplicity and affordability. However, these tests can yield false positives, especially in animals exposed to cross-reacting antigens or those with chronic infections [7, 8]. In contrast, molecular diagnostic methods, particularly polymerase chain reaction (PCR), offer higher specificity and sensitivity and have been increasingly used in epidemiological studies of brucellosis [9, 10].
Given the potential role of indigenous dogs in the maintenance and transmission of B. melitensis, this study aimed to assess the prevalence of infection in herding and stray dogs in the rural and urban areas of Neyshabur. Additionally, we sought to compare the diagnostic performance of serological and molecular methods in identifying infected animals.

2. Materials and Methods
2.1 Sample collection
This cross-sectional study was conducted on a total of 100 indigenous dogs from Neyshabur and surrounding rural areas of Razavi Khorasan Province, Iran. The sampled dogs were classified into two groups: herding dogs (n=50), which were in regular contact with livestock, and stray dogs (n=50), which roamed freely in peri-urban areas. Dogs were selected randomly, without consideration of age, sex, or clinical history, to provide a representative population sample.
Blood samples were collected aseptically from the cephalic or jugular vein of each dog. For molecular and bacteriological analysis, 2 mL of whole blood was drawn into ethylenediaminetetraacetic acid (EDTA)-containing tubes. For serological testing, 3 mL of blood was collected into plain tubes and allowed to clot. Serum was separated by centrifugation at 3,000 rpm for 10 minutes. All samples were stored at −21 °C and transported under cold-chain conditions to the laboratory for further analysis.

2.2 Serological test
Serological screening was initially performed using the RBPT following standard procedures. Briefly, a drop of serum was mixed with a drop of Brucella A and M antigen on a clean glass plate and gently agitated. The presence of visible agglutination within 4 minutes was considered a positive result; the absence of clumping was interpreted as negative [11]. To confirm RBPT-positive or equivocal results, the Wright standard tube agglutination test was carried out. A control tube was included in each set for comparative interpretation. Agglutination results were evaluated based on the clarity of the supernatant: a clearer supernatant compared to the control indicated a positive reaction, while a turbid or opaque supernatant was considered negative. In addition, the 2-mercaptoethanol (2-ME) test was performed on all positive and suspected samples to detect IgG antibodies and differentiate between acute and chronic infections. Results from all serological tests were recorded and compiled for final comparative analysis [7, 11-14].

2.3 Culture of blood samples
Blood samples were cultured in selective Brucella broth medium (Oxoid, Basingstoke, UK), supplemented with a combination of growth enhancers and selective antibiotics, including polymyxin B (2,500 IU), bacitracin (12,500 IU), cycloheximide (5,000 mg), nalidixic acid (250 mg), nystatin (50,000 IU), and vancomycin (1,000 mg). The cultures were incubated at 37 °C in an atmosphere containing 10% carbon dioxide for five days. Following incubation, samples were subcultured onto selective Brucella agar and incubated under the same conditions for an additional seven days.
Colonial morphology was examined, and suspected colonies were subjected to preliminary biochemical identification using catalase, oxidase, urease, nitrate reduction, hydrogen sulfide (H2S) production, and carbohydrate fermentation (glucose and lactose) tests [15]. Rose Bengal antigen derived from Brucella abortus was also used for agglutination testing, as it reacts with most Brucella species except Brucella canis [16]. Classical biotyping of isolates was performed based on the criteria established by Alton et al. [12], including assessment of CO₂ dependency, H₂S production, agglutination with monospecific A and M antisera, growth in media containing thionin and fuchsin dyes, agglutination with acriflavine, and susceptibility to lysis by the brucellosis-specific phage Tb (supplied by the Razi Institute, Iran). Biotyping results were interpreted according to established taxonomic standards in the literature.

2.4. DNA extraction
The extraction of genomic DNA from blood samples was performed with the MBST DNA extraction kit (made in Iran), following the instructions of the supplier. The quality and yield of extracted DNA were determined using the Nanodrop spectrophotometric method and confirmed by agarose gel electrophoresis. DNA samples were stored at −20 °C for later molecular analysis.

2.5. PCR test for identifying the genus and species of Brucella
The PCR was performed utilizing a master mix that was ready-to-use from Viragen (Amplicon, Denmark). The reaction mixture comprised 25 µL, with 12.5 µL of master mix, 1 µL of each of the primers (10 pmol/µL), 3 µL of DNA template, and 7.5 µL of nuclease-free distilled water [6, 17]. The primers and the conditions for the PCR cycling are provided in Table 1.

Distilled water served as the negative control, while a reference strain of B. melitensis (M16), obtained from the microbial collection of the Faculty of Veterinary Medicine, University of Tehran, served as the positive controls.
Amplification was conducted utilizing a 512-TC thermocycler (Techne, UK). PCR products were separated on a 2% agarose gel at 90 V in 1X TBE buffer for 70 minutes utilizing a 100 bp DNA ladder (Synaclone, Cat. No. 901644PR [7031SL]) for estimation of fragment size. The gels were stained with ethidium bromide (C7721MR) at 1 µg/mL, and the bands were observed under UV illumination (Bioword, Germany). 
Data were analyzed using SPSS software, version 26.0 (IBM Corp., Armonk, NY, USA). The prevalence of Brucella infection was calculated as the percentage of seropositive and PCR-positive cases among the total number of animals tested. Descriptive statistics were used to summarize categorical variables (e.g. sex, dog type). The chi-square test was used to assess the association between variables such as sex, dog type (herding vs stray), and infection status. A P<0.05 was considered statistically significant.

3. Result
3.1 Serological findings
The RBPT identified 31 seropositive dogs, representing an overall apparent seroprevalence of 31.0%. The highest proportion of seropositive animals was detected in herding females (50%), followed by stray females (40%), stray males (28.6%), and herding males (25%) (Table 2, Figure 1).

These results suggest widespread exposure to Brucella spp. across both stray and herding dog populations.

3.2 Bacteriological isolation and biotyping
Blood cultures from RBPT-positive samples were incubated on Brucella-selective agar. Colonies appeared after one week and were characterized by smooth, convex, grey morphology and an absence of hemolysis. Growth was not observed on MacConkey agar. Biochemical tests showed positive catalase, oxidase, urease, and nitrate reduction activity, with negative hydrogen sulfide (H₂S) production.
All isolates grew in the presence of basic fuchsine and thionin and were CO₂-independent. No lysis occurred with the Brucella-specific Tb phage. Classical biotyping identified all isolates as B. melitensis Biovar 1, based on agglutination with M-specific antiserum and standard phenotypic profiles [18].

3.3 Molecular detection by PCR
PCR analysis was conducted on all 31 seropositive blood samples. Using genus-specific primers (B4/B5), 3 samples (9.7%) yielded a distinct 223 bp product, confirming the presence of Brucella DNA. All three positive samples originated from herding dogs, indicating likely occupational exposure through close contact with infected livestock (Figure 2).

Subsequent testing of the genus-positive samples with B. melitensis-specific primers (Br.m) produced a clear 731 bp amplicon in all cases, confirming B. melitensis at the species level (Figure 3).

No amplification was detected in stray dogs or RBPT-negative controls.

3.4. Statistical associations
Statistical analysis revealed no significant association between PCR-confirmed infection and the sex of the dogs (χ²=0.09, df=1, P=0.76). In contrast, a significant association was found between infection and dog type: herding dogs were significantly more likely to test positive by PCR than stray dogs (χ²=6.21, df=1, P=0.013). Fisher’s exact test was applied for contingency tables with expected cell counts less than 5.

4. Discussion
Brucellosis is a chronic and widespread zoonotic disease caused by bacteria of the genus Brucella, presenting a persistent global threat to both human and animal health. Endemic in many regions, including parts of the Middle East, it remains challenging to detect, control, and eradicate due to complex transmission dynamics involving ecological, occupational, and animal–human interface factors [1, 3, 19]. Identifying local reservoirs and understanding species-specific transmission pathways are therefore critical components of brucellosis surveillance and control strategies.
In this study, we identified B. melitensis Biovar 1 in indigenous dog populations from Neyshabur and its surroundings, with PCR confirmation in 9.7% of RBPT-seropositive cases. Notably, molecular positivity was confined to herding dogs, underscoring the role of close, repeated contact with livestock, particularly infected sheep and goats, as a risk factor for cross-species transmission. This finding was consistent with prior evidence that Brucella spp. can infect non-preferred hosts through prolonged exposure [5, 20].
Despite relatively high RBPT seropositivity in stray females (40%) and males (28.6%), none of these animals tested positive via PCR. This disparity reflects the limited specificity of serological assays, particularly in stray populations that might have encountered environmental antigens or unrelated gram-negative bacteria. By contrast, PCR-confirmed prevalence among herding females (10%) and herding males (5%) supported the conclusion that occupational exposure, rather than incidental contact, plays a key role in the transmission of B. melitensis to dogs.
The findings of this study are aligned with those of Alamian and Dadar (2020), who reported B. melitensis in 38.1% of seropositive herding dogs from Tehran, Qom, and Alborz provinces using PCR, with 6 dogs also culture-positive [5]. The higher detection rate in their study compared to our 9.7% PCR-positive rate might reflect regional variation in livestock infection rates, diagnostic sensitivity, or differences in herd management practices. They also confirmed the presence of both Biovar 1 and 2, indicating a broader spectrum of circulating strains.
International studies corroborate our epidemiological observations. In Brazil, Keid et al. (2015) found a B. canis seroprevalence of 20.9% in 753 dogs, with no significant association between sex and infection status [21], a pattern that was mirrored in our results (P=0.76). Conversely, a statistically significant association between Brucella infection and dog type (P=0.013) highlighted herding dogs as a high-risk group due to continuous exposure to infected ruminants.
Age-related risk was not directly assessed in our study, but Mosallanejad et al. found a higher prevalence of B. canis antibodies in older dogs in Ahvaz, suggesting that duration and intensity of exposure, as seen in working dogs, might be more critical than age alone [18]. Similarly, Behzadi et al. (2011) reported 10.62% B. canis seropositivity in imported dogs in Shiraz [22], whereas our findings pointed to local transmission of B. melitensis in native, non-imported dogs, emphasizing differing transmission cycles for each Brucella spp.
The diagnostic superiority of PCR was also supported by Aras et al. (2011), who demonstrated its efficacy in post-mortem detection of B. canis in lymphatic tissue [23]. Our study confirmed that PCR is a reliable, non-invasive, and field-applicable method for detecting Brucella in canine blood samples, especially when combined with species-specific primers and sequence confirmation.
Serological assays such as RBPT, Wright, and 2-ME remained useful for preliminary screening but were subject to cross-reactivity and low specificity, especially in endemic areas. Known cross-reactants include Yersinia enterocolitica, Salmonella spp., Campylobacter fetus, and Vibrio cholerae, potentially inflating false positive rates [7, 9, 10, 15, 20, 24]. False negatives also occurred during early or chronic stages of infection, or in immunocompromised hosts [19, 20]. In our study, only 3 of 31 RBPT-positive dogs (9.68%) were PCR-confirmed, underscoring the limited predictive value of serology alone and reaffirmed the necessity of molecular diagnostics in brucellosis surveillance.

5. Conclusion
This study highlighted the role of herding dogs in Neyshabur as potential reservoirs of B. melitensis due to their close contact with infected livestock. While serological tests like RBPT aided in initial screening, their limitations, such as cross-reactivity and false positives, emphasized the need for more accurate diagnostics. PCR offered distinct advantages by enabling rapid, sensitive, and specific detection, even in asymptomatic or atypical hosts. The confirmed presence of B. melitensis in native herding dogs signals an ongoing risk to both animal and human populations. Effective control requires integrating molecular diagnostics into surveillance, vaccinating livestock, and educating animal owners. Lessons from countries with successful control programs showed that combining these measures could significantly reduce transmission and support long-term eradication efforts, especially in endemic regions like Iran.

Acknowledgements
The authors like to thank all of their colleagues at the Department of Clinical Sciences, Babol Branch, Islamic Azad University, Babol, Iran and Faculty of Veterinary Medicine, University of Tehran, Iran, for their kind cooperation.

Compliance with ethical guidelines
The authors declare that all ethical standards related to animal health and welfare have been respected in the present study.

Data availability
The data supporting the findings of this study are available upon request from the corresponding author.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization and study design: Fatemeh Zahra Gharib and Iradj Ashrafi Tamai; Data acquisition: Behnam Entezari, Fatemeh Zahra Gharib, and Iradj Ashrafi Tamai; Writing: Hesamoddin Ahmadi Afzadi and Iradj Ashrafi Tamai; Supervision: Fatemeh Zahra Gharib; Final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

References

1. Cutler SJ, Whatmore AM, Commander NJ. Brucellosis--new aspects of an old disease. J Appl Micr 2005; 98(6):1270-81. [DOI:10.1111/j.1365-2672.2005.02622.x] [PMID]
2. Whatmore AM, Davison N, Cloeckaert A, Al Dahouk S, Zygmunt MS, Brew SD, et al. Brucella papionis sp. nov., isolated from baboons (Papio spp.). Int J Syst Evol Microbiol. 2014; 64(Pt 12):4120-8. [DOI:10.1099/ijs.0.065482-0][PMID]
3. Ibrahim A, AbdelAll A, Amin A. Long-term diagnostic studies for detection of Brucella spp. in milk samples. Glob Vet. 2012; 8(1):54-61. [Link]
4. Moradi G, Esmaeilnasab N, Ghaderi E, Sofi Majidpour M, Salimzadeh H. Brucellosis in Kurdistan Province from 1997 to 2003. Ann Al-Quds Med. 2006; 2(1):32-7. [Link]
5. Alamian S, Dadar M. Brucella melitensis infection in dog: A critical issue in the control of brucellosis in ruminant farms. Comp Immunol Microbiol Infect Dis. 2020; 73:101554.[DOI:10.1016/j.cimid.2020.101554][PMID]
6. Baily GG, Krahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg. 1992; 95(4):271-5. [PMID]
7. Gall D, Nielsen K. Serological diagnosis of bovine brucellosis: Review of test performance and cost comparison. Rev Sci Tech. 2004; 23(3):989-1002. [DOI:10.20506/rst.23.3.1545][PMID]
8. Makarem E, Karjoo R, Omidi A. Frequency of Brucella melitensis in southern Iran. J Trop Pediatr. 1982; 28(2):97-100. [DOI:10.1093/tropej/28.2.97][PMID]
9. Doosti A, Dehkordi PG. Application of real-time PCR for identification and differentiation of Brucella abortus and Brucella melitensis in cattle. Bulg J Vet Med. 2011; 14(2):109-15. [Link]
10. Ocholi RA, Kwaga JK, Ajogi I, Bale JO. Phenotypic characterization of Brucella strains isolated from livestock in Nigeria. Vet Microbiol. 2004; 103(1-2):47-53. [DOI:10.1016/j.vetmic.2004.06.012][PMID]
11. Ilhan Z, Solmaz H, Aksakal A, Gülhan T, Ekin İH, Boynukara B. Detection of Brucella melitensis DNA in the milk of sheep after abortion by PCR assay. Arch Med Vet. 2008; 40(2):141-6. [DOI:10.4067/S0301-732X2008000200005]
12. Alton GG, Jones LM, Angus RD, Verger JM. Techniques for the brucellosis laboratory. Paris: INRA Publications; 198 [DOI: 10.1016/0007-1935(90)90017-W] [PMCID]
13. Ebid M, El Mola A, Salib F. Seroprevalence of brucellosis in sheep and goats in the Arabian Gulf region. Vet World. 2020; 13(8):1495-509. [DOI:10.14202/vetworld.2020.1495-1509][PMID]
14. Ghorbani A, Rabbani Khorasgani M, Zarkesh-Esfahani H, Sharifiyazdi H, Dehghani Kashani A, Emami H. Comparison of serology, culture and PCR for detection of brucellosis in slaughtered camels in Iran. Comp Clin Pathol. 2013; 22(5):913-7. [DOI:10.1007/s00580-012-1499-1]
15. Dadar M, Alamian S, Behrozikhah AM, Yazdani F, Kalantari A, Etemadi A, et al. Molecular identification of Brucella species and biovars associated with animal and human infection in Iran. Vet Res Forum. 2019; 10(4):315-21. [DOI:10.30466/vrf.2018.89680.2171][PMID]
16. Gafarpour M, Kotra Ebrahimi N, Nazemi A, Assmar M, Khataminezhad M. [Comparison of serological tests (Rose Bengal, Tube Wright and Coombs Wright) with molecular test (PCR) for detection of brucellosis (goat and sheep) (Persian)]. J Compar Pathobiol 2011; 7(4):337-42. [Link]
17. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol. 1994; 32(11):2660-6. [DOI:10.1128/jcm.32.11.2660-2666.1994][PMID]
18. Mosallanejad B, Ghorbanpoor Najafabadi M, Avizeh R, Mohammadian NE. A serological survey on Brucella canis in companion dogs in Ahvaz. Iran J Vet Res. 2009; 10(4):383-6. [DOI:10.22099/ijvr.2009.1731]
19. Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan P. Veterinary microbiology and microbial disease. 2nd ed. Oxford: Wiley-Blackwell; 2011. [Link]
20. Sadeghi H, Tamai IA, Vodjgani M, Gharagozlou F, Salehi TZ. Isolation and identification of Brucella melitensis biovar 1 using bacteriological, serological and molecular tools from Saanen goats in Alborz, Iran. J Vet Res. 2022; 77(2):fa107-15. [Link]
21. 20 Keid LB, Diniz JA, Oliveira TM, Ferreira HL, Soares RM. Evaluation of an immunochromatographic test to the diagnosis of Canine Brucellosis caused by Brucella canis. Reprod Domest Anim. 2015; 50(6):939-44. [DOI:10.1111/rda.12612][PMID]
22. Behzadi MA, Mogheiseh A. Epidemiological survey of Brucella canis infection in different breeds of dogs in Fars Province, I Pak Vet J. 2012; 32(2):234-6. [Link]
23. Aras Z, Uçan UŞ. Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR. Theriogenology. 2010; 74(4):658-62. [DOI:10.1016/j.theriogenology.2010.03.023][PMID]
24. Sharma D, Bhardwaj B, Chouhan H, Chandel B, Joseph B, Barolia S. Isolation and molecular characterization of Brucella melitensis from abortion storms in sheep with its zoonotic implications. Int J Curr Microbiol App Sci. 2018; 7(10):1348-56. [DOI:10.20546/ijcmas.2018.710.150]