Detection of Mycobacterium paratuberculosis in raw cow’s milk using polymerase chain reaction (PCR) technique

Paratuberculosis or Johne’s disease is a chronic debilitating disease mainly infects ruminants and caused by Mycobacterium paratuberculosis. Previous serological studies in Mosul city confirm the presence of positive reactants for paratuberculosis in cattle. However, culture methods to confirm the disease need a long incubation period and also special media. Raw cow’s milk is considered as potential source for transmission of M. paratuberculosis in cows’ herds. Accordingly, this study aimed to detect the presence of M. paratuberculosis specifically in the raw cow’s milk using polymerase chain reaction (PCR) technique as a rapid, sensitive and reliable method. A total of 50 samples of raw cow’s milk were collected from cows suffering from emaciation and unresponsive to antibiotic treatment. All the samples were subjected to DNA extraction and direct amplification PCR. The results showed that 3 (6%) out of 50 milk samples were positive for M. paratuberculosis. This is the first study in Mosul city that confirms the presence of M. paratuberculosis in raw cow’s milk using PCR technique. In conclusion, raw cow’s milk could be an important source for M. paratuberculosis infection in dairy cows, and also PCR technique could be helpful in rapid diagnosis of paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic wasting disease, which mainly infects ruminants and can frequently, be found in dairy cattle (1,2). The disease has worldwide distribution and leads to reduce milk yield of the affected herds and contribute to economic loss (2)(3)(4). The bacteria are mainly shedding through feces of the infected cattle (5). However, the milk also considers as an important source of infection for the other animals (6). Many studies are confirming the role of raw milk and colostrum as a potential source of infection (6,7). The gold standard method for diagnosis of paratuberculosis is bacterial culture. However, this method is cumbersome and timeconsuming, and cultured samples may need up to 3 months to consider negative (7). Therefore, the diagnosis is mainly relied on serological (8-10) and molecular methods (6,7,11,12). Paratuberculosis has been confirmed previously in several studies in Iraq. A previous preliminary study in Mosul city, Iraq confirmed the presence of seropositive antibodies against M. paratuberculosis in different ruminant's species including sheep (10), goats (13) and cattle (9) using enzyme-linked immunosorbent assay (ELISA). Another recent study in 2018 by Al-Farwachi and colleagues (14) confirm the presence of antibodies against M. paratuberculosis by ELISA in both diarrheic and nondiarrheic or apparently healthy cattle that brought to the Veterinary Teaching Hospital of College of Veterinary Medicine, Mosul University, Mosul, Iraq. Molecular methods offer rapid sensitive techniques for the diagnosis of paratuberculosis. Hence, the use of polymerase chain reaction as a molecular method can help in rapid detection of M. paratuberculosis in different types of samples (6,7,15,16), and offer a rapid diagnosis of the disease. Accordingly, this study aimed to detect the presence of M. paratuberculosis in raw cows' milk in Mosul city using PCR technique.

Sample collection
Total of 50 samples of raw cow′s milk was collected from cows suffering from emaciation, unresponsive to antibiotic treatment and retreating at Veterinary Teaching Hospital, College of Veterinary Medicine, University of Mosul, during the period of May to July 2018. A volume of 10 ml of milk was collected in a sterile test tube and transported to the Research Laboratory, Department of Veterinary Public Health, College of Veterinary Medicine. The samples were stored at -20 °C until being used for the DNA extraction process. For the preparation of samples, 10 ml of each milk sample was centrifuged at 3000 ×g for 15 min. The supernatant was discarded and the pellet was resuspended in 100 μl of saline solution (17).

DNA extraction
Mycobacterium paratuberculosis DNA was extracted using a commercial DNA extraction kit (Mycobacterium paratuberculosis Vet, Ref: VET-36, Sacace Biotechnology, Italy). According to manufacturer instructions, 1.5 ml Eppendorf tubes were prepared according to the number of tested samples. Each tube contains50 μl of milk sample, 20 μl of internal control and 50 µl of negative control of extraction. Additionally, 50 μl of negative control was added to a separate tube as negative control. 300 μl of lysis solution was added to each tube and vortexed and incubated for 5 min at 65 °C. All tubes were centrifuged for 30 sec at 8000 ×g. The supernatant was carefully removed without disturbing the pellet. After that, 300 μl of washing solution 1was added to each tube, then vortexed vigorously and centrifuged for 30 sec at 8000 ×g. The supernatant was discarded and 500 μl of washing solution 2 was added to each tube and vortexed vigorously and centrifuged for 30 sec at 8000 ×g. The supernatant was discarded and the pellet was resuspended in 50 μl of DNA-eluent and incubated for 5 min at 65 °C and vortexed periodically. After that, the tubes were centrifuged for 1 min at a maximum speed of 16000 ×g, and the supernatant contains purified DNA was kept at -20 °C for PCR assay.

Amplification and gel electrophoresis
The amplification of the target M. paratuberculosis gene was performed according to the manufacturer instruction (Mycobacterium paratuberculosis Vet, Ref: VET-36, Sacace Biotechnology, Italy). Briefly, PCR-mix-1tubes were prepared and 10 μl of PCR-mix-2 was pipetted into each PCR-mix-1 tube. Then, 10 μl of extracted DNA was added to the corresponding tubes. For the negative control tube, 10 μl of DNA buffer was added. Additionally, for positive control tube, 10 μl of positive control (diluted 1:10 with DNA buffer) was added. The PCR was performed using Eppendorf Thermocycler (Eppendorf, Germany) with one cycle at 95 °C for 2 min, 42 cycles consisting of (step 1: 95 °C for 1 min, step 2: 66 °C for 1 min, step 3: 72 °C for 1 min). Then, one cycle at 72 °C for 1 min was set for final extension. Finally, the reactions were cooled at 10°C until proceeding to the gel electrophoresis. Analysis of results was based on the presence of specific bands of amplified gene using 2% agarose gel stained with ethidium bromide (Amersham Bioscience, USA), and visualized using UV transilluminator and digital camera. The presence of specific amplified DNA fragment with209 bp indicating a positive result for M. paratuberculosis, in addition, internal control with a product size of 700 bp must also appear to ensure that the extraction was valid.

Results
The results showed that only 3 out of 50 milk samples were positive and the incidence rate was 6%. PCR products with a size of 209 bp were detected in all positive samples. In addition, internal control bands were also detected with product size of 700 bp, indicating successful DNA extraction as shown in Figure (1).

Discussion
Paratuberculosis has gained great importance worldwide due to severe economic losses (3,4). The disease mainly affects ruminants including cattle, sheep and goats, and causes chronic contagious enteritis that characterized by unresponsive to treatment (18,19). Recent studies in Mosul city referred to the presence of seropositive reactant against M. paratuberculosis in sheep, goats and cattle which give evidence of the importance of the disease in ruminant (9,10,13,14). In this study, M. paratuberculosis was detected in (6%) 3/50 of tested cow's milk, which indicates direct shedding of the pathogen in the raw milk. Since paratuberculosis is a chronic disease and clinical signs may only appear after years of infection, the secretion of M. paratuberculosis in milk is considered as main risk factor for transmission and spreading of the disease to calves (6,11,20,21). In addition, the zoonotic potential of M. paratuberculosis via consumption of contaminated milk and milk products is of public health importance due to frequent isolation of M. paratuberculosis from Crohn's disease patients (22)(23)(24)(25). The PCR used in our study offer sensitive and rapid method for detection and confirmation of the presence of M. paratuberculosis in clinical samples (6,7,11). Unlike, culture method that requires special media and experienced personnel, PCR might be conducted in few hours in the equipped laboratory, while culture method might need several months to consider negative results (6,7,25,26). The PCR technique allowed the detection of fewer M. paratuberculosis cells in 2 mL of milk, making this procedure is rapid, sensitive and cost-effective for the diagnosis of clinical and subclinical paratuberculosis (11,27). In a study targeting cattle population in south of Iraq 29/81 (35.8%) were positive for M. paratuberculosis using PCR from buffy coat of seropositive cows detected by ELISA, and this highlighted the importance of PCR as a confirmatory tool (28). Another study in Brazil, M. paratuberculosis was detected in 20 (16.5%) out of 121 of milk samples using conventional PCR (29). Another study in Cyprus confirms the presence of M. paratuberculosis in 63 (28.6%) positive samples out of 220 milk samples collected from milk storage (25). However, in our study the lower number of positive samples compared with these studies may be due to sporadic cases that have been received in our Veterinary Teaching Hospital rather than detection of infected farms.

Conclusion
In conclusion, M. paratuberculosis was detected successfully in raw cow's milk using PCR which indicates the significance of milk as a potential source of infection in dairy cows.