Detection of Brucella Spp. in Raw Milk and Dairy Products of Traditional Domestic Dairy Sale Centers by Real-time PCR in Semnan

1. Introduction
Brucellosis is among the most widespread zoonotic diseases in Iran, with profound public health and economic consequences [1]. In humans, the disease affects various tissues and organs and can be categorized as acute, sub-acute, or chronic forms, often leading to prolonged illness and disability [2]. Brucella melitensis and Brucella abortus are the most pathogenic species for humans and can infect a wide range of domestic animals, including, cattle, buffalo, goats, sheep, and camels. In animals, brucellosis is associated with abortions, stillbirth, weak calves, genital infections, placentitis, epididymitis, and orchitis, with bacteria excreted through uterine discharges and milk. Human infection occurs primarily via the consumption of raw or unpasteurized dairy products or through direct contact with infected animals. Brucella species can survive in raw milk for 10 days, in fresh cheese for up to 3 months, and in ice cream and cream for some time [3]. Foodborne diseases are an important public health problem as they not only affect human health, but also have a significant impact on economic and trade issues. Studies linking different pathogens in food to the disease in humans would help quantify the risk of foodborne diseases. Moreover, the prevention of foodborne diseases in general is a complex effort, involving many different actors along the chain of production from the farm to food service [4, 5]. Globally, the World Health Organization (WHO) estimates approximately 500000 new human cases annually. 
Despite vaccination campaigns and control measures, brucellosis remains endemic in parts of Middle East, South America, and Asia. In Iran, surveys suggest that about 7.4% of cattle are infected. Transmission continues largely through the informal distribution of raw dairy products, particularly in rural communities with limited awareness of hygienic practices [6]. Conventional diagnostic methods, such as culture, are time-consuming, hazardous, and often yield false negatives due to the fastidious nature of the pathogen. Serologic methods, while rapid, may lack specificity because of cross-reactivity and insufficient antibody levels. Molecular techniques, particularly polymerase chain reaction (PCR) and real -time PCR (qPCR), provide superior sensitivity, specificity, and biosafety. 

1.1. Real-time PCR [7, 8]
The use of qPCR has provided several advantages over conventional PCR, such as quantification, real-time, and in-situ analyses, in addition to automation. In this technique, the PCR products are detected as they accumulate, and the amount of generated PCR product is proportional to the increase in a fluorescent signal, which is monitored during the exponential phase. This technique the permits rapid identification and quantification of bacteria [9].
The present study aimed to investigate the presence of Brucella spp. in raw milk and traditional dairy products sold in Semnan, Iran. Given the social, economic, and geographical conditions of Semnan, where most people use traditional milk products, there is a possibility of people contracting this disease. Using real-time PCR with SYBR Green dye, we targeted genus-specific genes to detect B. abortus and B. melitensis. The findings provide insights into the prevalence of brucellosis in local dairy products and inform strategies for food safety and disease control. 

2. Material and Methods
2.1. Samples preparation
A cross-sectional study involving three consecutive sampling rounds was conducted over approximately three months (early spring to late spring 2020) on traditional dairy products sold in Semnan Province, Iran. Based on a previously reported average prevalence of 6.6% for similar products, a 95% confidence level (Z=1.96), and a 5% margin of error, the sample size was calculated using Cochran’s formula, yielding 95 samples [6]. The total number of samples was proportionally allocated to different dairy products according to their market sales volume, resulting in 50 raw milk samples, 23 traditional ice cream samples, 11 traditional cream samples, and 11 unpasteurized cheese samples, all collected randomly from traditional dairy sales centers.

2.2. DNA extraction
For each sample, 300 µL was processed using the Dyna Bio DNA Mini Kit (Takapouzist Co., Iran) according to the manufacturer’s instructions. The purity and quantity of extracted DNA were measured using a NanoDrop spectrophotometer (Thermo Scientific Nanodrop, Wilmington, USA) at 260 and 280 nm (A260/280 ratio). Extracted DNA was eluted in 50 µL elution buffer. Primers targeted the bcsp31 gene (223 bp fragment) for genus-level detection. Species-specific primers targeted a 498 bp fragment (B. abortus, alkB gene) and a 731 bp fragment (B. melitensis, BMEI 1162 gene). The specifications of the primers used are listed in Table 1.

The primers’ specificity was assessed using BLAST [10].

2.3. Real-time PCR procedure
Amplification was performed using a Rotor-Gene (Q MDx, Germany). The amplification reactions contained 2 µL DNA template, 2.5 U AmpliTaq Gold DNA polymerase (Ampliqon, Copenhagen, Denmark), 1X (5 µL of 10X) GeneAmp buffer II, 6 mM MgCl2, 800 μM GeneAmp dNTP blend (BioFact, Copenhagen, Denmark), 300 nM of each primer, and sterile water. The cycling conditions utilized in this experiment were as follows: an initial denaturation step was conducted at 95 °C for a duration of 10 minutes. This was succeeded by 40 cycles, which included denaturation at 95 °C for 10 seconds, primer annealing at 55 °C for 30 seconds, and extension at 72 °C for 30 seconds. Subsequently, a melting curve analysis was executed over a temperature range of 65 °C to 95 °C, with a transition rate of 0.1 °C/s, based on continuous fluorescence measurements. The positive control contained the Brucella strain ATCC 23456, while the negative control contained nuclease free water. PCR efficiency was evaluated by constructing a standard curve from serial dilutions of quantified Brucella control DNA. Cycle threshold (Ct) values were plotted versus log10 initial copy number. The slope of the regression line was used to calculate efficiency based on Equation 1:

1. E=10−1/slope−1 [11, 12]. 
The limit of detection (LOD) was evaluated using the Brucella strain ATCC 23456, the standard strain was cultured in BHI broth overnight at 37 °C and then adjusted to a turbidity equal to 0.5 McFarland standard tube (0.08–0.1 absorbance at 600 nm wavelength). Then, eight standards (S1–S8) were prepared by 10-fold serial dilutions (from 1 to 0.00000001) of the adjusted BHI broth (equal to 0.5 McFarland). Genomic DNA was extracted from all standards, and quantitative real-time PCR was performed. The log linear phase of the reaction was used to determine the cycle threshold (Ct) for each standard. To obtain a reference line, the Ct values of the eight standards with known numbers of bacteria (S1=107; S2=106; S3=105; S4=104; S5=103; S6=102; S7=10; S8=1 bacteria per mL, respectively) were determined as previously described by Kralik and Ricchi. The linear regression equation obtained from the reference standard was employed to quantify Brucella spp. in genomic DNA, with the findings represented as log 10 CFU/g (colony-forming units per gram) [11, 12].

2.4. Statistical analysis
Differences in Brucella prevalence among dairy product types were assessed using chi-square and Fisher’s exact tests. Bacterial load (CFU/g or mL) between products was compared using the nonparametric Kruskal–Walli’s test, followed by the Mann–Whitney U test for pairwise comparisons. A P<0.05 was considered statistically significant.

3. Results
The real-time PCR technique was assessed through the analysis of the Ct value, melting temperature (Tm), specificity, sensitivity, and efficiency of the standard curve. 

3.1. Molecular identification of Brucella spp. in milk and traditional dairy products using real-time PCR method
Among 95 samples analyzed using real-time PCR, four were positive for Brucella spp.; specifically, three contained B. abortus, and one sample in raw milk contained B. melitensis (Table 2).

No significant overall difference in Brucella prevalence was found across products (P=0.093), and bacterial load analysis revealed higher median CFU in cheese (>10⁷) compared to milk and ice cream (P=0.067).

3.2. Sensitivity and specificity of real-time PCR      
To precisely pinpoint the target genes in Brucella spp. and determine the optimal annealing temperature for the primer sets in real-time PCR, we conducted a series of PCR reactions. The outcomes of these reactions were visualized through electrophoresis on a 1% agarose gel (Figure 1).

The standard curve exhibited excellent linearity (R²> 0.99) between log₁₀ DNA concentration and Ct values. The slope was −3.26, corresponding to a reaction efficiency of 102%, which is well within the optimal range (90–110%) for qPCR assays (Figure 2).

The melting (Figure 3) and proliferation curves (Figure 4) for both positive samples and controls, utilizing targeted primers for the bcsp31 gene, alkB, and BMEI1162, are presented to support the identification of the Brucella spp.

and their relevant species (Figure 5).

Results from the real-time PCR analysis demonstrated that three samples tested positive for B. abortus (alkB gene), while one sample was confirmed as B. melitensis (BMEI1162 gene) among the four positive detections.

3.3. Quantification of Brucella Spp. DNA concentrations in positive samples using a standard curve 
The proliferation and melting curves for the dilutions derived from both the standard and positive samples are illustrated in Figure 6.

It shows the drawn melting curve resulting from the reactions in the dilutions prepared from the positive control. These curves, which are all in the same temperature range, show the accuracy of the manufactured products and, as a result, the accuracy of the test. 

3.4. The equation concentration of DNA in positive samples with the bacterial counts (CFU/gram or Cc)
A comparative analysis, performed utilizing a NanoDrop ND-1000 UV-Vis’s spectrophotometer (Thermo Scientific NanoDrop, Wilmington, USA) as outlined in Table 3, reveals a significant correlation between CFU/mL and the yield of DNA obtained from standardized dilutions.

This correlation is quantitatively represented by the equation derived from the standard curve depicted in Figure 7.

Consequently, the quantification of DNA extracted from the positive samples was executed based on their positions along this standard curve, and the results are summarized in Table 4.

Standard curves demonstrated strong linearity between DNA concentration and bacterial counts (R2 >0.99). The DNA concentration of positive samples ranged from 0.72 to 62.94 ng/ µL, corresponding to bacterial loads of 1-107 CFU/mL. Cheese rined samples showed the highest bacterial counts (>107 CFU/g).

4. Discussion
The detection of Brucella spp. in 4.21% of samples highlights the ongoing risk of brucellosis transmission through raw and traditional dairy products in Semnan, Iran. The higher prevalence in cheese (18.18%) compared with the milk and ice cream suggests that certain traditional products may provide favorite conditions for bacterial persistence.
Our findings align with previous studies in Iran, where contamination rates varied wildly depending on product type, region, and diagnostic methods. For examples, studies have reported contamination rates of 2-10% in milk and up to 18% in cheese. Differences likely reflect variations in sample size, livestock vaccination coverage, and hygienic practices. Internationally, prevalence has been reported as high as 40% in some African regions, while many European countries have achieved elimination through strict control programs.
This disease can be transmitted to humans through contaminated dairy products or contact with infected animals [13-15]. 
Multiple investigations in Iran have explored the presence of B. abortus and B. melitensis in raw cow’s milk and dairy products to assess potential risks for consumers. In a study examined 238 unpasteurized dairy products from Shiraz province. 5.04% of the products were contaminated. Contamination was found in 18.75% of raw milk and 6.25% of yogurt samples. Cheese, dough, and traditional ice cream samples were contamination-free. Some of the contaminated samples contained B. abortus, B. melitensis, or both [16,17]. A study in Sarab, East Azerbaijan, Iran, found that 2.2% of 1000 cheese samples tested positive for B. melitensis and B. abortus. Similarly, research conducted in Toisarkan, Hamedan, Iran, identified 21 milk samples contaminated with B. abortus and 18 samples with B. melitensis. Additionally, in Isfahan and Chaharmahal and Bakhtiari, Iran, screenings revealed the existence of 1% B. abortus in raw cow’s milk, local cheese (comprising 2.5% B. abortus, B. melitensis), and traditional cream (containing 1% B. abortus); in contrast traditional ice cream samples showed no contamination [18,19]. 
A semi-nested PCR method was employed to identify the presence of Brucella bacteria in samples of raw milk and cheese, revealing differing levels of contamination. The rates of Brucella contamination in various dairy products were as follows: 45.5% in raw goat milk, 39.1% in unpasteurized cheese, 27.3% in raw sheep milk, 26.3% in raw cow milk, 25% in pasteurized cheese, and 14.7% in pasteurized milk [16]. A study in Kurdistan, Iran, by Shafei et al. (2012) reported that 33.33% of 60 raw cow’s milk samples were infected with Brucella spp., with 45% of those samples being B. abortus [19]. The prevalence of Brucellosis in Kurdistan was associated with its proximity to neighboring countries like Iraq and Turkey, leading to the introduction of non-native strains. Positive Brucella spp. samples were also identified in milk samples through PCR in Kerman, Iran. Another study in Lorestan, Iran, in 2017 found a 10% prevalence of Brucella spp. in 120 milk samples. Brucella spp. are found in raw milk in Sudan and Kenya with prevalence rates of 22.4% and 40%, and 18.9% and 65.5%, respectively. In Iraq, the prevalence of Brucella spp. infection varies between 8.4% and 56%, as determined through blood testing. European countries have successfully eliminated Brucellosis or have kept the disease prevalence low. Discrepancies in results from these studies can be attributed to methodological differences, sample sizes, regional factors, livestock vaccination, and disease control measures [20-24]. 
Recent studies have shown that PCR is more sensitive and accurate in detecting Brucella spp. in various food items compared to traditional methods. One study found that 75% of samples tested positive for B. abortus, suggesting a high utilization of cow’s milk in dairy production. Increased consumption of milk and dairy products can elevate the risk of Brucellosis, with research indicating that the infectious dose for Brucella spp. in food products ranges from 10 to 100 CFU/g/mL. The SYBR Green Real-Time PCR method used in a recent study could detect less than one CFU/g/mL of the product, demonstrating higher accuracy and sensitivity than previous methods [24]. In Semnan, Iran, B. abortus and B. melitensis have been detected in raw milk and traditional dairy products, indicating their presence in the local livestock populations. Regions that consume unpasteurized milk are at risk of Brucella spp. transmission, which poses a significant public health concern. Real-time PCR method with Sayber Green dye is advised for its precision and efficiency in detecting Brucella spp. During the spring and summer months, 75% of the samples analyzed showed the presence of B. abortus and B. melitensis, aligning with the periods when infected animals are breeding and lactating. Using techniques like real-time PCR for Brucella spp. detection in dairy products can improve pathogen identification accuracy. Public health strategies should emphasize the vaccination of livestock, strict control of raw milk sales, and consumer education about the risks of unpasteurized dairy consumption. Seasonal monitoring is also critical, as higher prevalence often coincides with breeding and lactation periods.

Acknowledgements
We hereby extend our gratitude to the Research Council of Semnan University for the support of this study.

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Data availability
The data that support 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, study design, and supervision: Mahnoosh Parsaeimehr; Sample collection and execution of tests: Ali Nademirad and Hamid Staji; Data analysis and data interpretation: Hamid Staji and Ashkan Jebellijavan; Statistical analysis: Ashkan Jebellijavan; Writing: Mahnoosh Parsaeimehr and Marzieh Heidarieh; Writing: Mahnoosh Parsaeimehr and Marzieh Heidarieh; Final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

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