Molecular detection of Mycoplasma spp. from camel’s milk

1-    Mycoplasma spp. was widely distributed in camel’s milk samples.

2-    M. bovis are considered one of the bacteria that cause subclinical mastitis.

3-    The PCR method is regarded as a simple, rapid, method for detecting the Mycoplasma bacteria in camel’s milk samples.

Introduction

The dromedary camel, or Arabian one-humped camel, is scientifically known as Camelus dromedarius and is found in many countries in Asia and Africa, especially those with desert and semidesert climate (1-4). Camels are essential because they are used as a means of transport in the desert and consider one source of economic income that comes from wool, milk, and meat production (1,5,6) and from using camels in racing (7,8). Camel’s milk considers an exceptional nutritional food because of its high nutritional and therapeutic uses (9-13). Although camels consider more resistant to many diseases that occur in other animals (14), many diseases can affect camels and lead to crucial economic loss (15). In many countries, including Iraq, camel’s milk production was affected by mammary gland infection, also known as mastitis (16). Clinical mastitis may be associated with the change in milk colour and composition associated with swelling, painful mammary glands, and/or remaining without any apparent signs of subclinical mastitis (16-20). Many etiological agents can cause camels mastitis, including Escherichia coli, Staphylococcus spp, Streptococcus agalactiae, Micrococcus spp, Arcanobacterium spp, Mannhiemia spp. Salmonella spp, Klebsiella spp. (21),and finally, Mycoplasmas spp. (15,22). Mycoplasma spp. was a cell wall deficient prokaryotes bacteria classified under the class Mollicutes, and its definitive as the smallest organism capable of self-replicating (14). Most of the Mycoplasma infections in camels remain undiagnosed, partly because of the scarcity of camel's Mycoplasma studies (23) and because the Mycoplasma classical diagnosis method is complex and takes a long time. Moreover, the Mycoplasma serological tests could give false results because of the cross-reactivity of Mycoplasma spp. with other closely related bacterial species (14, 24,25). In recent decades, molecular methods have been used to diagnose Mycoplasma infection. The introduction of molecular diagnoses using the polymerase chain reaction (PCR) method with a specific primer provides a rapid and accurate tool to detect and identify Mycoplasma infections (14,15,26-29). The previous studies reported that many species of Mycoplasma were associated with mammary gland infection in cows, including M. Bovis, M. californium, M. bovigenitalium, M. alkalescens, and M. canadense (30). M. bovis is the most widely contagious species that spreads rapidly throughout the herd (31) that causing eye, joint, respiratory, or urogenital infection (32) and is mainly associated with M. bovis mastitis (18).

Although camels are an essential source of zoonotic diseases such as the Middle East respiratory syndrome and other bacterial diseases like Mycoplasmosis (7,33), very few articles focus on Mycoplasma mastitis, especially that caused by M. bovis in camels in Iraq and the Arabic area. The current study aimed to detect Mycoplasma's prevalence, especially M. bovis in camel’s milk.

Materials and methods

Ethical approval and Data collection permit

      All milk sample was collected after taking approval from the she-camel owners for sampling, all animals were treated ethically during the milk sampling, and according to the authorized ID. UM. Vet. 2022.013 which was provided by the Mosul University/college of veterinary medicine/Institutional Animal Care and Use Committee.

Sampling

Fifty milk samples were collected from one-humped camels distributed in the Badia Al Jazeera area in Al- Anbar and Nineveh province from February to June 2022 (Figure 1). The samples were collected from She-Camels that were suffering from subclinical mastitis. A 20 ml of each milk sample was collected in a sterile test tube and transported under the cooling condition (in a cool box containing Co₂ ice) to the Department of Microbiology and Department of Veterinary Public Health, College of Veterinary Medicine, Mosul University. All milk samples were saved in deep freeze until used for DNA extraction.

Figure 1: Geographical distribution map of camel milk sample.

Mycoplasma DNA extraction

The DNA of the genus Mycoplasma. was extracted from milk samples by using the gSYNC™ Geneaid extraction kit (Geneaid Biotech Ltd, Taiwan) according to company instructions. Two primer pairs were used: a universal primer set to detect the genus Mycoplasma and a second specific-species primer for detecting M. bovis. All the primers were synthesized and supplied by Bioneer cooperation / Korea, according to the sequence noted by (34,35), to produce amplicon sizes of 285 bp and 232 bp, respectively (Table 1).

Table 1: Primers used to detect genus Mycoplasma and M. bovis

The DNA amplification was done by using a 25 μl mixture containing (5.5 μl dd distal water, 12.5 μl 2.5X PCR master mix, 1 μl from both forward and reverse primer, 5 μl extracted mycoplasma DNA, 1.5 μl MgCl₂); the amplification programs was set as describer (Table 2). All the PCR products were migrated in 1.5% agarose (Biometra/Germany), and DNA bands were visualized by a UV transilluminator (Biometra /Germany) and photographed.

Table 2: The amplification programs used to detect the genus Mycoplasma and M. bovis

* Temperature used for amplification of Mycoplasma genus. ** Temperature used for amplification of the specific-species gene of M. bovis.

Results

Our study shows that 26.66% (13/50) of camel’s milk samples were infected with Mycoplasma spp. In addition, the prevalence rate of M. bovis from milk samples was high and reached 61.53% (8/13) from all positive milk Mycoplasma infections, the result showed that total isolation for M. bovis from all milk samples 16 %(8/50).

The amplification of PCR product in 1.5% agarose gel reveals one high specific band in 258 and 232 bp for Mycoplasma and M. bovis respectively (Figures 2 and 3).

Figure 2: Amplified PCR products of Mycoplasma Gene (M: Molecular base ladder, 1: Positive control, 11: Negative control, 2-10: milk sample)

Figure 3: Amplified PCR products of M. bovis gene (M: Molecular base ladder, 1: Positive control, 9: Negative control, 2-8: milk sample)

Discussion

Camel’s milk shows great economic importance nowadays because of the long lactating period of camels with the high nutrient values and therapeutical uses of its milk (9), so any disease in the mammary gland that affects milk production and causes significant loss is associated with zoonotic infection and cost of animal’s welfare (36). Many microorganisms can grow and multiply in camel’s milk, especially after entering the teat channel, which comes from either the teat/mammary gland surface, milker hand, or the environment that causes camel's mastitis (37-39). Bacterial camel's mastitis can be either clinical and/or subclinical. The subclinical mastitis was most crucial as it did not show any clinical signs and did not cause any noticeable change in the udder or milk, farther that subclinical mastitis comes before clinical mastitis and may take a long period to diagnose, which causes the animals' source for environmental contamination and spread of disease for other animals (40,41). Many studies focus on camel's mastitis and show that about 25% were subclinical cases in Ethiopia (12), 16.6% in Saudi Arabia (42), and 11.7% in the United Arab Emirates (43). Finally, 25.8% in Egypt (41) were associated with different bacterial isolates (17,37). This percentage was near to that recorded in our study, reaching about 26% from all milk samples, which indicates highly spread of Mycoplasma infection in milk.

In our country, very few articles were published dealing with camel's mastitis, and no research about the role of Mycoplasma bacteria in camel’s mastitis was recorded. Our study firstly isolated M. bovis from camel’s milk with a high percentage reaching 61.5% of the total milk sample. This indicates that M. bovis can be the primary source of subclinical camel's mastitis infection. M. bovis was globally spread and able to cause many diseases, including pneumonia, keratoconjunctivitis, abortion, infertility, and mastitis (32,44,45). Most mammary gland infection caused by M. bovis was subclinical and associated with a respiratory infection. Mahmoud et al. (46) reported that M. bovis was found in the respiratory tract in both diseased and healthy camels in Egypt (46). Iraqi articles focus on the isolation of other bacteria rather than Mycoplasma spp. from mastitis camel’s milk which used conventional bacteriological methods which not suitable to diagnose Mycoplasma as it takes a long time to grow this many explain partially leaks of Iraqi articles dealing with camel's Mycoplasma mastitis. Unlike our result, Tigani et al. (47) was unable to diagnose M. bovis in camels' gangrenous mastitis by PCR in their study conducted in the United Arab Emirates (47). This may be due to some PCR inhibiter in a sample, like sampling time and /or contamination with other bacteria, which lead to false-negative results (35). Only 61.53% of Mycoplasma milks M. bovis caused the infection, indicating that other Mycoplasma species may cause subclinical mastitis in camels. This agrees with (22) how reported isolation of M. arginine from camel sub-clinical mastitis in Sudan.

Conclusion

This was the first local study in Iraq that detected the ability of Mycoplasma, especially M. bovis to cause subclinical camel mastitis by using the PCR method as the primary diagnostic molecular method.

Acknowledgement

The authors appreciate the support and effort that foreword from the College of Veterinary Medicine, the University of Mosul that assisted in the achievement of this manuscript.

Conflict of interest

There was no conflict of interest.