Introduction

Camelus dromedaries or Arabian camels are important livestock animals living in semiarid and arid environments (1-5); camel's number reached 12.5 million in 2003, according to WHO (1). In Iraq, Camels are one of the essential demotic animals in a Bedouin desert area and play a significant economic as a source of milk, wool, meat, and skin production (5-8). Camel's milk is an extraordinary food with high nutritional value and has therapeutic uses (5). camel milk is also considered a potent antioxidant (4); camels resist many diseases (9). However, many camels may be affected with mammary gland infection (mastitis), which may occur as clinical mastitis or subclinical mastitis, associated with pain, swelling of mammary glands with composition, and milk color changes (10-14). Many bacterial species can cause mastitis in camel, including many types of Enterobacteriaceae (5); camel mastitis is predominantly caused by Escherichia coli (E. coli), a Gram-negative rod-shaped bacterium. Notably, E. coli is commonly present in the intestinal tracts of both animals and humans (15). Escherichia coli can be classified into different pathotypes based on their virulence, pathogenicity, and site of infection. These pathotypes include commensal, enteropathogenic, and extraintestinal strains (16-20). Many E. coli is armed with virulence factors that enable them to survive in their host and environment (21,22). E. coli virulence factors may include capsule, adhesin properties, and production of toxins; many E. coli is considered intestinal pathogenic and divided into eight groups according to their infectivity and virulence (23-25). The Shiga toxin-producing E. coli (STEC), which contain Shiga toxins (Stx1 and Stx2), have foodborne importance as a primary causative agent of hemorrhagic colitis, hemolytic uremic syndrome, and bloody diarrhea; both these toxins encoding in locus for enterocyte effacement (LEE) pathogenicity island which that contain another gene as eae A which less pathogenic than Stx1 and Stx2 which responsible for severe infection in animals (26,27). The Stx1 and Stx2 genes produce many toxin subtypes; the Stx1 toxin includes three subtypes, while Stx2 has nine subtypes (28). Both the Stx1 and Stx2 toxins may cause food poisoning in humans after consuming raw milk contaminated with STEC, resulting in various signs and symptoms depending on the virulence of the Stx toxin subtypes (29,30).

In many cases, camel milk can be consumed unpasteurized due to its therapeutic worth (31) and rich nutritional value (5). This led to becoming an essential source of zoonotic disease; for this reason, this study aimed to isolate E. coli and detect Shiga toxins genes (Stx1 and Stx2) in isolates.

Materials and methods

Sampling

In the present study, conducted between February and May 2023, 50 milk samples were obtained from female one-humped camels exhibiting subclinical - mastitis using the California Mastitis Test. The collection was conducted in the Badia Al Jazeera area of Al-Anbar and Nineveh province. Each sample, comprising 20 ml of milk, was obtained using sterile containers. To maintain sample integrity, they were transported in a cold box with CO₂ ice to the central Laboratory under cooling conditions.

Ethical approval

All samples were collected following owner approval, and the study was carried out based on the ethical guidelines provided by the Institutional Animal Care and Use Committee at Mosul University's College of Veterinary Medicine, with an authorized ID of UM.Vet.2023.016.

Isolation and identification of E. coli

The milk samples were examined to separate and identify pathogenic strains of E. coli. To do this, all samples were placed in nutrient broth (LAB, United Kingdom) and incubated for 24 h at a temperature of 37°C. To follow the classical method, a single loopful of the nutrient broth was spread onto Eosin Methylene Blue Agar (EMB) and MacConkey agar (LAB, United Kingdom). These plates were then incubated for 24 h at 37°C. Additionally, Brilliance E. coli/coliform Agar (Oxoid, United Kingdom) was employed to differentiate between generic E. coli and coliform bacteria. To confirm the presence of suspected E. coli isolates, various biochemical tests were conducted, including Gram staining, Indole testing, Methyl Red testing, Citrate Utilization testing, Voges-Proskauer testing, as well as Catalase, Oxidase, and Triple Sugar Iron agar (32). To preserve the E. coli isolates, they were stored in Nutrition broth (15% glycerol) and kept at -80ºC until further use.

DNA Isolation

The following procedures were performed to isolate and analyze doubtful E. coli isolates. First, the samples were grown on Brilliance E. coli/coliform media and incubated for 24 h at 37°C. This specific agar medium facilitates the growth and differentiation of E. coli and coliform bacteria. To isolate the DNA of E. coli, the DNeasy Blood and tissue kit from Geneaid (Korea) was utilized following the instructions. This kit provides a reliable method for extracting DNA from various sources, including bacteria. The isolated DNA was then quantified using the Bio-drop device, allowing for accurate DNA concentration estimation. Finally, the isolated DNA of E. coli was stored at -20°C to maintain its stability and quality for subsequent analyses. This ensures that the DNA remains preserved and ready for further investigation and experiments.

Amplification of uid A, stx1, and stx2 Genes

The PCR method was conducted to amplify the uidA, Stx1, and Stx2 sequences of the isolated E. coli bacteria, as indicated in table 1. The PCR reaction was performed in a total quantity of 25 μl. The reaction mishmash consisted of 12.5 μl of 2× GoTaq Green Mix Master (Promega Corporation, USA), 1 μl of primer 1, 1 μl of primer 2, 6.5 μl of DNeasy-free water (Promega Corporation, USA), and 4 μl of the E. coli DNA template. Subsequently, the amplicons of the target sequences were visualized through gel electrophoresis. A 1.5% agarose gel (Peqlab, Erlangen, Germany) was prepared, and the DNA samples were loaded into wells along with a DNA marker (100 bp ladder). Electrophoresis was then performed, allowing for the separation and visualization of the amplified DNA fragments, which were compared to the DNA ladder for size estimation. The mixture was added to an Eppendorf tube, and the total volume was adjusted to 25 μl. The PCR amplification uses appropriate thermal cycling conditions. The specific thermal cycling conditions would depend on the PCR protocol, including the denaturation, annealing, and extension temperatures and durations. These conditions are typically optimized for each primer set, and the DNA template is amplified.

Table 1: The sequence Primers and PCR program used for detecting the uidA, stx1, and stx2 gene

PCR program: I=35 times (94°C – 30s, 57°C – 30s, 72°C – 30s), II=35 times (94°C – 30s, 55°C – 30s, 72°C – 30s)

Results

The conventional microbiology diagnosis for camel’s milk reveals isolation of 33/50 E. coli isolate, all E. coli isolate was emphasized using specific uid A gene that gives 632bp amplicon specific for genus E. coli (Figure 1), with a total isolation rate reaching 60% from all subclinical mastitis. Further analysis of E. coli isolates to detect Stx1 and Stx2 genes show that positive PCR amplicons (347 bp and 592 bp), respectively, some isolates carrying either Stx1 and /or Stx2 with positive occurrence reach 63% (21/33) and 27% (9/33) for Stx1 and Stx2 respectively (Figures 2 and 3).

Figure 1: Length of Gene Sequences in Base Pairs: uidA (623 bp), M Lane: DNA ladder, 1 Lane: Negative Control, 2-6 Lanes: Positive E. coli isolates, 1 Lane: Positive Control (Shiga Toxin-Producing E. coli)

Figure 2: Length of Gene Sequences in Base Pairs: Stx1 (347 bp), M Lane: DNA ladder, 1 Lane: Negative Control, 2-6 Lanes: Positive E. coli isolates, 1 Lane: Positive Control (Shiga Toxin-Producing E. coli)

Figure 3: Length of Gene Sequences in Base Pairs: Stx2 (592 bp), M Lane: DNA ladder, 1 Lane: Negative Control, 2-6 Lanes: Positive E. coli isolates, 1 Lane: Positive Control (Shiga Toxin-Producing E. coli)

Discussion

Like other dairy animals, camel mastitis is considered a severe disease that affects the quantity and quality of milk and increases the cost of treatment, leading to economic loss. Although the diagnosis of clinical mastitis could be made quickly with some tests, relatively little research has been focused on camel mastitis, particularly those caused by bacteria. Subclinical mastitis required more significant attention to confirm the health and safety of camels, particularly in desert areas (35). Many research showed the ability to isolate E. coli in subclinical mastitis as the main causative agent (36). The present result showed that E. coli isolates, which PCR confirmed, reached 66% of all milk samples, which was higher than 3% in Oman (37), 7% in Saudi (38), and 36% in Egypt (39). In addition, the results of this study were near to the results of the previous study in Kenya, which found the percentage of E. coli isolated from camels' milk was 56.5% (40). The higher isolate percentage in the current study may be related to the fact that subclinical mastitis occurred in higher prevalence than clinical mastitis and required more time to be detected (35), farther than most information for subclinical bacterial mastitis depends on milk bacteriological examination and show that Enterobacteriaceae was predominant in mastitis (37,41); this lead to different bacterial type and isolation percentage comes from different research that can cause either clinical or subclinical mastitis.

The result showed that E. coli isolated harboring 63% and 27% for Stx1 and 2, respectively; many research listed different percentages for either Stx1 and 2 from different sources. Onlen et al. (42) reported that the percentage reached 15.6 % and 6.3% for Stx2 and Stx1, respectively, in E. coli isolated from food; other research shows a higher percentage of Stx2 and Stx1 in E. coli isolated from dairy cattle which reach to 100% and 93.1% respectively (43) different result showed by other research includes 15% and 13% for Stx2 and Stx1 genes, respectively from human isolate (44). In comparison, Bulgarian cattle show 9.08% and 4.54% for Stx2 and Stx1 (45); in camel milk, the frequency of Stx2 and Stx1 may reach 77.7, 46.2 (39), while, another study found that 23.1% of E. coli isolates possessed the Stx1 gene and no one of these isolates have the Stx2 gene (40). This study agreed with Njage et al. (40) that the difference in virulence gene occurrence is closely related to animal species and sources of E. coli isolated. Diab et al. (39) mention that Stx1 was a frequently detected virulence gene among E. coli camel isolate, which comes compatible with the present results.

Conclusion

2. coli causes serious illness in camels and results in subclinical mastitis that may affect the quality and quantity of milk and result in economic and public health problems; our isolated E. coli harbor many different virulence genes with a higher percentage of Stx1 than Stx2.

Acknowledgment               

The authors greatly thank University of Mosul, College of Veterinary Medicine for providing facilities to achieve this study.

Conflict of interest

There is no conflicting interest.