Introduction

Mycoplasma bovis is a bacteria classified as belonging to the family Mycoplasmataceae, and it is characterized by a small genome, a lack of a cell wall, and high nutritional requirements for in vitro growth (1). Mycoplasma bovis has been isolated for the first time from a case of epidemic mastitis in cows in 1961 in the state of California in America (2). In terms of risk, it is classified in List B, which infects cows and calves (3). It is highly adapted in ruminants, especially cattle and even humans, who have continuous contact with infected cows (4). Mycoplasma bovis can be spread between animals in herds either directly or indirectly; direct transmission between cows can occur during milking or through nose-to-nose contact, but indirect transmission can also occur through shared drinking and feeding troughs (5). Furthermore, it spread from calves suffering from respiratory illnesses and cows with clinical mastitis, suggesting that the pathogen may spread from dairy cows to their young through contaminated milk (6). Several infectious diseases in cattle, including bovine mycoplasmosis, are caused by M. bovis in calves and cows (6,7). These diseases include vasculitis and keratoconjunctivitis (8), otitis media and decupital abscesses (9), endocarditis, which has been documented (10), chronic bronchopneumonia, polyarthritis, contagious mastitis (11), subclinical mastitis (12), and abortion and genital problems (13). Depending on the severity of clinical signs, the disease's morbidity rate might range from 20 to 80%, or its mortality rate could range from 3 to 50% as a result of persistent chronic pneumonia and secondary bacterial infections (14). Mycoplasma bovis is globally spread, especially in North America, Australia, Europe, and Asia (15). It was detected in cows and calves infected with pneumonia, polyarthritis, and mastitis in Nineveh Governorate, Iraq, using an indirect enzyme-linked immunosorbent assay (i-ELISA) (16). Moreover, using the PCR technique, Hamad et al. (17) diagnosed M. bovis in calves infected with pneumonia in Mosul City, Iraq. There are different genes of M. bovis that have been used for the detection of M. bovis, such as uvrC and gapA (18, 19), 16S rRNA, oppD, and oppF genes (20), gyrA, gyrB, and parC genes (21,22), polC and 16S-23S rRNA ITS genes (23,24), ma-mp81 and mb-mp81 genes (25,26), and gltX gene (27). Moreover, Okella et al. (28) stated that uvrC is the most commonly used gene, followed by oppD and 16S-23S rRNA ITS gene. Numerous laboratory methods have been used to detect M. bovis in infected cattle, such as isolating bacteria and elector microscopy (7). Additional serological tests are employed, including indirect ELISA (29), immunohistochemistry (IHC) (23), and the polymerase chain reaction (PCR) technique (30). Due to the problematic processing, which was time-consuming, and the identification of Mycoplasma at the genus level only using the culture method, further cross-reactivity of M. bovis with other pathogens was observed in the serological tests (28). Therefore, PCR techniques are the most commonly used for the specific and rapid detection of M. bovis (31,32).

This study aimed to determine the prevalence of M. bovis in cattle using PCR techniques and, for the first time, investigate the phylogenetic analysis of M. bovis in Nineveh governorate, Iraq.

Materials and methods

Ethical approval

The institutional animal care and use committee in the College of Veterinary Medicine, University of Mosul, ethically permitted this study (UM.VET. 2022.085) on February 15, 2022.

Animals and sampling collection

This study was conducted on 352 cattle of different ages, sexes, origins, and regions of Nineveh Governorate. During the period from March 2022 to February 2023, various samples, including blood sample (n=352), nasal swab (n=352), ocular swabs (n=40), synovial fluid (n=65), and milk sample (n=30), were randomly collected from 352 cattle (14, 30,33-35). These samples were kept at -20°C until performed for the conventional PCR (c-PCR) and Multiplex PCR (m-PCR) techniques to detect Mycoplasma spp. and M. bovis, respectively.

DNA extraction for Mycoplasma detection

The DNA of Mycoplasma spp. was extracted from all the above samples using the commercial PrimePrep Genomic DNA Extraction Kit from tissues (GeNetBio, Korea). The DNA was extracted according to the manufacturer's instructions. Using the Nanodrop (BioDrop, Germany), the concentration of extracted DNA was estimated at wavelength 260nm, while the purity of extracted DNA was assessed by calculating the ratio of (A260 nm to A280 nm) as described by Morais et al. (36).

Amplification of the Mycoplasma DNA

The c-PCR technique was used to amplify the 16S ribosomal RNA (16S rRNA) gene of Mycoplasma spp., in cattle's nasal swabs using ‘universal’ primers; M.Genus-F (5’-GGG AGC AAA CAC GAT AGA TAC CCT-3’) and M.Genus-R (5’-TGC ACC ATC TGT CAC TCT GTT AAC CTC-3’), that was designed by Botes et al. (37). In addition, the m-PCR technique was used to amplify the deoxyribodipyrimidine photolyase (uvrC) gene and glyceraldehyde-3-phosphate dehydrogenase GAPDH (gapA) gene of Mycoplasma bovis in cattle cultured nasal swabs that positive in the c-PCR technique, also the nasal swab samples negative to the c-PCR technique. using specific primers: uvr-F(5’-TTA CGC AAG AGA ATG CTT CA-3’), uvr-R (5’-TAG GAA AGC ACC CTA TTG AT-3’), that was designed by Subramaniam et al. (38) and gap-F (5’-ATA GGA GGA TCC AAA AGA GTC GCT ATC AAT GGT TTT GGA CG-3’), gap-R (5’-GGA AAT GGT ACC TTA CTT AGT TAG TTT AGC AAA GTA TGT TAA TG-3’), that was designed by Perez-Casal and Prysliak, (39), respectively. All these primers were provided by Macrogen Inc., South Korea. The positive bands were at approximately 270bp, 1626bp, and 1007bp. For 16S rRNA, uvrC, and gapA genes, respectively. Furthermore, a clinically and laboratory-positive cow's DNA was used as a positive control, while all PCR components except DNA were used as a negative control.

The PCR reactions for detecting the Mycoplasma spp. and M. bovis were performed in a total volume of 25μl for each one. Furthermore, the thermocycler was set with some modifications in steps according to Botes et al. (37), Subramaniam et al. (38), and Perez-Casal and Prysliak (39) (Table 1). The Midori green-stained (Axon Scientific Sdn Bhd, Malaysia) and 1.5% agarose gel were used to electrophorese the PCR products and to visualize the resultant bands, UV transillumination (BIO-RAD/USA) was utilized.

Table 1: The PCR program for DNA samples subject to conventional PCR and multiplex PCR techniques

Sequencing and phylogenetic analysis of Mycoplasma bovis DNA

The DNA PCR products (n=15) for the uvrC gene out of 108 positive samples of M. bovis in cattle from various samples, including blood sample (n=3), nasal swab (n=3), ocular swabs (n=3), synovial fluid (n=3), and milk sample (n=3). Furthermore, the DNA products (n=15) for the gapA gene out of 101 positive samples of M. bovis in cattle from various samples, including blood sample (n=3), nasal swab (n=3), ocular swabs (n=3), synovial fluid (n=3), and milk sample (n=3), were sent to Macrogen Inc., South Korea for sequencing. The retrieved uvrC and gapA genes local sequences were analyzed using different online programs such as NCBI Blastn (to determine the similarity between obtained sequences and other sequences in the NCBI GenBank), multiple sequence alignments (CLUSTALW) program (to determine the alignment scores (within and between) obtained sequences), and BankIt tool program (to deposited five sequences for each uvrC gene and gapA gene of M. bovis in the NCBI GenBank). Moreover, MEGA11 software was used to create phylogenetic trees with the outgroup sequence (U00089.2)-Mycoplasma pneumoniae strain M129, Germany (40,41).

Statistical analysis

To calculate the prevalence of M. bovis, descriptive statistics on the Excel program 2010 was used, and to determine the significant differences between sample types and types of genes, an X-Square 2x2 table in IBM SPSS Version 22 (Inc., Chicago, USA) was used. Values were considered significant at P<0.05.

Results

In this study using Nanodrop, the concentration of DNA extracted from the blood samples, nasal swabs, ocular swabs, synovial fluid, and milk samples ranged between 60.5 - 327.5 ng/µl, and the purity of the extracted DNA varied between 1.7 - 1.9. Based on the amplified 16S rRNA gene, the prevalence of Mycoplasma spp. was 38.63% (136 out of 352) in cattle cultured nasal swabs Nineveh Governorate using the c-PCR technique (Table 2), and the positive bands were approximately at 270 bp. (Figure 1). Further, there was no significant prevalence of M. bovis in the cattle's cultured nasal swabs, which was positive in the culture method. Also, the nasal swab samples were negative to the culture method based on amplified uvrC and gapA genes, which was 30.68% (108 out of 352) and 28.69% (101 out of 352 cattle), respectively, using the m-PCR technique, and the positive bands were approximately at 270 bp (Figures 1 and 2). In addition, the result also revealed that there was no statistically significant difference in the prevalence of M. bovis in various types of samples from 352 cattle that were examined using the m-PCR technique based on amplified uvrC and gapA genes, including blood samples, nasal swabs, ocular swabs, synovial fluid, and milk samples, which were 27, 30.68, 22.50, 27.69, and 23.33%, respectively (Table 3).

Table 2: Prevalence of Mycoplasma spp. and Mycoplasma bovis in cattle using conventional PCR and multiplex-PCR techniques using cultured nasal swab

Vertical different letters (a or b) means that the values are significantly different P<0.05. Number of samples testes 352 samples.

Figure 1: Conventional PCR technique detected 16S rRNA gene of the Mycoplasma spp. in approximately band size 270bp; Lane M) DNA ladder; Lane N) Negative control. 

Figure 2: Multiplex PCR technique detected uvrC and gapA genes of the Mycoplasma bovis in approximately band size 1626bp. and 1007bp. respectively; Lane M) DNA ladder; Lane P) Positive control; Lane N) Negative control. 

Table 3: Prevalence of Mycoplasma bovis based on the type of samples using conventional PCR technique

 Vertical different letters (a or b) means that the values are significantly different (P < 0.05)

In the present study, based on the multiple sequence’s alignment program, the alignment score within the local sequences of the uvrC gene (n=15) and gapA gene (n=15) was 98.41-100 and 98.83-100, respectively. In contrast, there was 23.12 - 25.78 between the sequences of uvrC and gapA genes (Table 4). In addition, for the first time, five sequences from different samples, one for each (blood sample, nasal swab, ocular swab, synovial fluid, and milk sample), out of 15 local sequences of the uvrC gene of M. bovis in cattle in Nineveh governorate, were deposited at the NCBI-American GenBank with the accession numbers OR784598.1, OR784599.1, OR784600.1, OR784601.1 and OR784602.1 (Table 5). Moreover, for the first time, five sequences from different samples, one for each (blood sample, nasal swab, ocular swab, synovial fluid, and milk sample), out of 15 local sequences of the gapA gene of M. bovis in cattle in Nineveh governorate, were deposited at the NCBI-American GenBank with the accession numbers OR792211.1, OR792212.1, OR792213.1, OR792214.1 and OR792215.1 (Table 5).

Table 4: Alignment score within and between local sequences of the uvrC and gapA genes of Mycoplasma bovis using multiple sequence alignment program

Table 5: The type of samples and the GenBank accession numbers of local sequences for uvrC and gapA genes of Mycoplasma bovis in cattle

In the present study, individual sequencing analysis for the local sequences (OR784598.1, OR784599.1, OR784600.1, OR784601.1, and OR784602.1) of the uvrC gene for M. bovis was observed to be highly similarity (99.81%–100% identity) to those sequences in the GenBank of various countries such as Canada (CP042938.1, CP042939.1), Iran (KX772803.1, KX772803.1, KP795974.1), Poland (KU168342.1), Switzerland (AF003959.1, LT578453.1), and Egypt (KP099618) using the NCBI Blastn program (Table 6). Furthermore, individual sequencing analysis for the local sequences (OR792211.1, OR792212.1, OR792213.1, OR792214.1, and OR792215.1) of the gapA gene for M. bovis was observed to be highly similar (99.13%–100% identity) to those sequences in the GenBank of different countries such as Canada (EF436267.1) and Egypt (KU559606, KP099616) using the same program NCBI Blastn (Table 7).

Table 6: Similarity between the local sequences of the uvrC gene for Mycoplasma bovis and other sequences of the same pathogen in the GenBank using NCBI BLASTn

Table 7: Similarity between the local sequences of the gapA gene for Mycoplasma bovis and other sequences of the same pathogen in the GenBank using NCBI BLASTn

The phylogenetic analysis of the local partial sequences of the uvrC for M. bovis OR784598.1, OR784599.1, OR784600.1, OR784601.1 and OR784602.1 revealed highly phylogenetic properties and an extremely close evolutionary relationship 80-99% to other global sequences of the same pathogen that have been recorded in the Genbank for different countries, such as Canada, Iran, Poland, Switzerland, and Egypt, after performing 1000 nucleotide sequence reconstruction using MEGA 11-Bootstrap analysis (Figure 3). In addition, the phylogenetic analysis of the local partial sequences of the gapA for M. bovis OR792211.1, OR792212.1, OR792213.1, OR792214.1 and OR792215.1 revealed highly phylogenetic properties and an extremely close evolutionary relationship 84-100% to other global sequences of the same pathogen that have been recorded in the Genbank for different countries, such as Canada and Egypt, after performing 1000 nucleotide sequence reconstruction using MEGA 11-Bootstrap analysis (Figure 4).

Figure 3: Phylogenic tree of the partial sequences of the uvrC gene of Mycoplasma bovis in Nineveh Governorate, Iraq (*), with the outgroup Mycoplasma pneumoniae strain M129 (U00089.2), Germany.

Figure 4: Phylogenic tree of the partial sequences of the gapA gene of Mycoplasma bovis in Nineveh Governorate, Iraq (*), with the outgroup Mycoplasma pneumoniae strain M129 (U00089.2), Germany.

Discussion

In the current work, the prevalence of M. bovis in cattle in Nineveh Governorate was 30.68% using the c-PCR technique. This finding was lower when compared with other previous reports that mentioned the prevalence of M. bovis in Iraq. Mahmood and Rhaymah (16) and Hamad et al. (17) stated that the prevalence of M. bovis in the calves in Mosul city was 76.09% and 86.5% using i-ELISA and PCR techniques, respectively. Furthermore, different studies indicated the prevalence of M. bovis in cattle in various countries using various diagnostic laboratory methods such as in Iran was 8.8% using nested PCR (n-PCR) technique (42), in Jordan was 27.3% using conventional PCR (c-PCR) technique (43), in Saudi Arabia was 24% using c-PCR technique (44), in Turkey was 23.3% using direct fluorescent antibody test (DFAT) (45), in China was 48.7% using i-ELSA (46), in Egypt was 67.5% and 8.3% using culture method and c-PCR technique respectively (26,47), in Sudan was 7.2% using i-ELISA (48), in United States of America was 100% and 87.5% using LAMP and real-time PCR (RT-PCR) technique respectively (27), in Brazil was 91.4%, 1.1% and 62.3% using (IHC), RT-PCR technique, and i-ELISA respectively (19,49,50), and in Australia was 42.5% using i-ELISA (51). The differs in the prevalence of M. bovis among counties may be due to different management approaches, environmental conditions, efficient diagnostic techniques used in other studies, the types of samples that were tested, and the presence or absence of additional factors, such as the host's age, physical characteristics, and immunological status (22,23,28,52-54).

In this study, there was no significant difference in the prevalence of M. bovis in cattle based on amplified uvrC and gapA genes, with alignment scores within each gene of 98.41-100 and 98.83-100 respectively. These results agree with Abdeen et al. (18), who stated that there was no significant difference in the prevalence of M. bovis based on uvrC and gapA genes using the PCR technique, and the similarity within each gene was 95.3% and 100%, respectively. The uvrC gene and gapA were selected for the PCR technique to detect M. bovis in cattle because they are the most commonly used in epidemiology, sequencing, and phylogenic analyses studies, and they are available in molecular databases (18,19,39,55,56).

The current study used PCR techniques to detect M. bovis in cattle. According to the types of PCR techniques, the sensitivity and specificity differed for the detection of M. bovis when compared with the culture method, such as 97.2% sensitivity and 90.9% specificity of the LAMP PCR technique and 86.1% sensitivity and 92.9% specificity of the c-PCR technique (57). Moreover, Parker et al. (1) and Scott et al. (32) noted that PCR techniques have greater efficiency, specificity, and sensitivity for laboratory detection of M. bovis.

In addition, the result showed no significant difference in the prevalence of M. bovis in various types of samples (blood samples, nasal swabs, ocular swabs, synovial fluid, and milk samples) when tested using the m-PCR technique. This result corresponds to Parker et al. (1), Clothier et al. (58), Jain et al. (59), Parker et al. (60), and Zhao et al. (61) they mentioned that there was no difference in the percentage of M. bovis among the types of specimens using different diagnostic tools. The interpretation for the reasons is that M. bovis is present in various secretions and causes different diseases in infected cattle, which explains why it can be isolated from multiple organs and samples (1,7,28).

Results concerning the sequencing and phylogenetic analysis of the PCR products (n= 15) for the uvrC gene and (n=15) for the gapA gene of M. bovis obtained from cattle's blood samples, nasal swabs, ocular swabs, synovial fluid, and milk samples, were sequenced. Five sequences from each gene were deposited in the NCBI GenBank OR784598.1-OR784602.1 and OR792211.1- OR792215.1 of the uvrC and gapA genes, respectively, for the first time in Nineveh governorate. These sequences were observed to have phylogenetic characteristics and a very tight evolutionary relationship with the other M. bovis sequences in the NCBI GenBank of different countries such as Canada (24,38), Iran (62,63), Poland (64), Switzerland (39,65), and Egypt (26,66), with high similarity 99.13%-100% after 1000 replications using MEGA11 software (41).

Conclusions

This study concludes that M. bovis is widely distributed and circulating among cattle in Nineveh governorate, Iraq. There was no significant difference between the uvrC and gapA genes, also among various samples, in detecting M. bovis using the m-PCR technique. Furthermore, sequencing and phylogenetic analysis of M. bovis play essential roles in the study areas' strategic control of M. bovis.

Acknowledgments

The authors thank the College of Veterinary Medicine, University of Mosul, for their support.

Conflict of interest

The authors claim no conflicts of interest.