287 Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 Accepted: 23.08.2021 | Available on line: 31.12.2022 1Faculty of Veterinary Medicine, Ahmadu Bello University P.M.B 1045 Zaria, Kaduna State, Nigeria. 2Faculty of Veterinary Medicine, University of Ilorin, P.M.B 1515 Ilorin, Kwara State, Nigeria. 3Faculty of Veterinary Medicine, University of Abuja, P.M.B 117 Gwagwalada. Abuja, Nigeria. 4 WOAH Reference Laboratory for Contagious Bovine Pleuropneumonia, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy. *Corresponding author at: : Faculty of Veterinary Medicine, Ahmadu Bello University P.M.B 1045 Zaria, Kaduna State, Nigeria. E-mail: markfrancis4u@gmail.com. Markus Isa Francis1, Mashood Abiola Raji2, Clara Nna Kwanashie1, Jibril Adamu1, Lushaikyaa Allam1, James Agbo Ameh3, Godwin Onyemaechi Egwu2, Katiuscia Zilli4, Flavio Sacchini4 and Massimo Scacchia4 Keywords Cattle, Contagious bovine pleuropneumonia, Mycoplasma mycoides subspecies mycoides, PCR-RFLP, Nigeria. Summary This study aimed to perform molecular typing of Mycoplasma mycoides subsp. mycoides from slaughtered cattle in Adamawa and Taraba States, north-eastern Nigeria. A total of four hundred and eighty (480) samples of lung tissues, nasal swabs, ear swabs and pleural fluids were collected from cattle at slaughter and processed according to standard laboratory protocols. Identification and confirmation were achieved with specific PCR and PCR-RFLP. An overall M.  mycoides subsp. mycoides isolation rate of 6.87% (33/480) was obtained. In Adamawa State, 12 (10.91%) isolates of M. mycoides subsp. mycoides came from both, lung tissues and pleural fluids. While in Taraba State, 5 (7.14%) and 4 (5.71%) isolates of M. mycoides subsp.  mycoides came from lung tissues and pleural fluids, respectively. The samples from nasal and ear swabs from the study states were negative for M. mycoides subsp. mycoides. Thirty-three out of the 37 culture positive isolates were confirmed to be Mycoplasma mycoides subspecies mycoides with the production of a band equivalent to 574-bp. Molecular typing with restriction endonuclease Vsp1 results in the two bands of 180-bp and 380-bp. In conclusion, the study has established an isolation rate of 6.87% for M. mycoides subsp.  mycoides. Measures to strengthen movement control in order to minimise the spread of this dreaded disease of cattle were recommended. Identification of Mycoplasma mycoides subspecies mycoides from slaughtered cattle in two transboundary states of North‑eastern Nigeria Mycoplasma mycoides subsp.  mycoides infects lung tissues and can be transmitted through inhalation of infectious aerosols of the organism by a susceptible animal (OIE 2018). Severe inflammatory, exudative lesions at lung and pleural membranes characterize the disease. In calves, however, infection of M.  mycoides subsp.  mycoides results mainly in arthritis and associated lameness, but rarely in pulmonary lesions (OIE 2015). Other factors such as movement of trade cattle, seasonal migration, transhumance and normadism also enhance the spread of the infection (Aliyu et al. 2000). The WOAH recommended complement fixation test (CFT), competitive enzyme-linked immunosorbent assay (c-ELISA) and polymerase chain reaction for the diagnosis of CBPP (OIE 2018). Isolation Introduction Mycoplasma mycoides subspecies  mycoides (M.  mycoides subsp.  mycoides) is an important bacterium pathogen of cattle causing contagious bovine pleuropneumonia (CBPP), which is one of the most severe endemic infectious disease in sub-Saharan Africa. The disease is causing major social and economical impact, due to the animal losses and international trade restrictions (Mariner et  al. 2006, Dupuy et  al. 2012, Fischer et  al. 2015, Parker et al. 2018). M. mycoides subsp. mycoides was the first Mycoplasma to be described, as extracellular bacterium that lives in close association with their host cells (Dupuy et  al. 2012). The bacterium lacks of cell wall, and has the capacity of self-replication (Westberg et al. 2004, Di Teodoro et al. 2020). 288 Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 M. mycoides subsp. mycoides in Nigeria Francis et al. border. Adamawa and Taraba States have a land area of about 91,390 km2 with tropical wet and dry climate. The two states are among the lead producers of livestock in Nigeria with an estimated cattle population of 3.5 million (Francis et al. 2018b). Sample collection and storage The samples used for this study were obtained from cattle slaughtered at abattoirs within the areas under study between 2016 and 2017. The samples were collected for a period of thirteen (13) months. A total of four hundred and eighty (480) samples of pneumonic suspected lung tissues (180), nasal swabs (180), ear swabs (60) and pleural fluid (60) were collected from 190 cattle heads at slaughter, when they were showing pathological lesions suspected for CBPP. Two hundred and eighty four (284) and one hundred and ninety six (196) samples from cattle were collected from the abattoirs in Adamawa and Taraba States, respectively (Table  I). All the collected samples from abattoirs in Yola and Jalingo were transported to the National Veterinary Research Institute Zonal Offices in Yola and Jalingo, respectively. Samples were processed in 2 ml sterile Nalgene® cryo vials containing 1.5 ml of pleuropneumonia-like organism (PPLO) broth (Difco) supplemented (horse serum, sodium pyruvate and fresh yeast extract) and were later transported in refrigerated Coleman box to the Mycoplasma Laboratory, National Veterinary Research Institute (NVRI) Vom, Plateau State and then stored at - 20 °C prior to further processing. and identification of mycoplasmas is still the gold standard even though it requires a well-equipped laboratory with expertise for these very fastidious and slow-growing organisms (McAuliffe et  al. 2005, Egwu et  al. 2012). The use of PCR for the rapid and specific identification of M. mycoides subsp. mycoides has its limitations in developing countries, but detection of these species by PCR proved to be highly efficient with better results than culturing techniques (Egwu et al. 2012, Wade et al. 2015). The disease threatens livestock production, limits international trade and is therefore of huge economic concern in affected countries (Fadiga et al. 2013). In Nigeria, estimated CBPP morbidity and mortality rate of up to 50% and 25%, respectively, have been documented with annual economic losses of more than 2.2 billion Nigerian Naira (Fadiga et al. 2013). Outbreaks of the disease still occur in the northern region which harbours three-quarter of the country’s 19.5 million cattle population (Tambuwal et al. 2011). Several epidemiological studies have been conducted in Nigeria to assess the situation of CBPP in the North-eastern region (Ameh et al. 1998, Aliyu et  al. 2000, Francis et  al. 2018a) but there was no report on the molecular typing of the causative agent. Recent information about the disease is scanty and no extensive work been done in the study area. The present study was aimed to perform molecular identification and typing of M.  mycoides subsp. mycoides from slaughtered cattle in Adamawa and Taraba States, North-eastern Nigeria, with the aim to help in clarifying the characteristics of the strains circulating in the study area. Materials and methods Study area Adamawa and Taraba States are located in the South-eastern part of Nigeria (Figure 1). They lie between latitude 7°0’0” and 11°0’0”N and between Longitude 9°0’0” and 13°0’0”E. The Yola Modern Abattoir in Adamawa State is located on the Latitude 9°13’33.3”N and Longitude 12°27’10.1”E. An average of 70-80 cattle is slaughtered daily in the abattoir, while numbers are higher during festivity period. Jalingo Abattoir in Taraba State is located on the Latitude 8°54’12.1”N and Longitude 11°21’14.3”E with an average of 40-50 cattle slaughtered daily. Adamawa State shares boundaries with Taraba, Borno, Gombe States while Taraba State is bounded by Gombe, Bauchi, Plateau, Nassarawa and Benue States. They both share an international boundary with the Cameroon Republic along the southeastern Figure 1. Map of Adamawa and Taraba State, North‑eastern Nigeria showing the study areas. 289Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 Francis et al. M. mycoides subsp. mycoides in Nigeria agarose gel, stained with ethidium bromide (van Kuppeveld et  al. 1994). This PCR was able to detect Mycoplasma spp. in general and it is routinely used to confirm the presence of mycoplasmas in samples. Isolates that yielded positive to Mycoplasma species underwent additional testing using a Mycoplasma specific PCR. Identification of Mycoplasma mycoides subspecies using PCR This protocol is a PCR-based test for the specific identification of M.  mycoides subspecies according to Bashiruddin and colleagues (Bashiruddin et  al. 1994). For molecular characterization, the nucleic acid of the samples were further amplified using a set of primers: MM450 (forward) 5’-GTATTTTCCTTTCTAATTTG-3’ and MM451 (reverse): 5’-AAATCAAATTAAGTTTG-3’ targeting the CAP-21 genomic region of 16S rRNA. An initial denaturation at 94 °C for 5 minutes was followed by 40 cycles at 94 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 30 seconds, and then a final extension at 72 °C for 5 minutes. Mycoplasma mycoides sub-clusters were used as positive controls and the expected positive PCR products of 574 bp were visualized on a 1% agarose (Musa et al. 2016). The PCR products were electrophoresed on 1% agarose gel and the samples run for 30 minutes. The resultant bands were stained with ethidium bromide and visualised under ultraviolet light (Bashiruddin et al. 1994). Differentiation of Mycoplasma mycoides subspecies mycoides using PCR‑RFLP The typing method was adapted from the protocol used in WOAH Reference Laboratory for CBPP and that of Bashiruddin and colleagues (Bashiruddin et al. 2005), but varied with respect to Vsp1 (8-12 U/µl concentration) which replaced Asn1 endonuclease in this study. The PCR-RFLP was performed according to the method described by Musa and colleagues (Musa et al. 2016). Results Out of the 480 samples tested, 39 (8.13%) were positive for Mycoplasma species by culture. In Adamawa State, 25 (8.80%) isolations were made from 12 (10.91%) lung tissues and 13 (11.82%) pleural fluid. In Taraba State, 8 (11.43%) lung tissues, 5 (7.14%) pleural fluid and 1 (3.57%) ear swab yielded a characteristic ‘fried egg’ colonies, typical of Mycoplasma, with an isolation rate of 7.14% (n = 14). The thirty nine (39) pure isolates of Mycoplasma Isolation of mycoplasmas The samples were processed according to the method described by Thiaucourt and colleagues (Thiaucourt et  al. 1992). The PPLO broth (Difco) supplemented, containing various samples were filtered by passing through 0.45 µm millipore filters (Millipore Merck, Burlington, MA, USA) which is permeable to organisms smaller than 0.45 µm in size. The filtrate was then incubated at 37 °C in screw-capped bottles containing supplemented PPLO broth (Difco) for three (3) days under 5% CO 2 and later cultured onto PPLO agar (Difco) supplemented. These were incubated at 37 °C in a 5% CO 2 atmosphere with maximum humidity, and examined daily for a period of 1-2 weeks for evidence of growth, using a stereomicroscope. Isolated colonies having the classical ‘fried eggs’ appearance with dense raised centres (nipples) were triple-cloned into 10 ml of PPLO broth (Difco), and incubated for one week. Both, broth and agar media were considered negative if no comet or Mycoplasma-like colonies were observed after 14 days of incubation. Cloned isolates were stored at + 4 °C. These isolates were later transported refrigerated at 2-8 °C by Express Courier to the WOAH Reference Laboratory for CBPP at the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZSAM), Teramo, Italy. Upon receipt, the isolates were stored at +  4  °C, verified and then registered under the exotic disease unit before being processed. Identification and confirmation of Mycoplasma mycoides subsp. mycoides isolates DNA extraction Deoxyribonucleic acid was extracted from 500  µl of PPLO broth presenting comet colonies using Maxwell® 16 Tissue/cell DNA Purification kits (Promega, Wincosin, USA) with Maxwell® 16  instrument (Promega, Wincosin, USA) according to the manufacturer’s instructions and the extracted DNA was kept at - 20 °C until analyzed. Identification of Mycoplasma species using specific PCR The nucleic acid from each sample was tested using a PCR specific for Mycoplasma species as previously described (van Kuppeveld et al. 1994). The Mycoplasma group-specific primer set which amplifies a 280-bp fragment, consists of the forward primer 5’-GGGAGCAAACAGGATTAGATACCCT-3’ and the reverse primer 5’-TGCACCATCTGTCACTCTGTTAACCTC-3’. The aliquots were analyzed by electrophoresis on 2% 290 Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 M. mycoides subsp. mycoides in Nigeria Francis et al. area were negative for M. mycoides subsp. mycoides (Table I). Thirty-three (33) out of the thirty-seven (37) Mycoplasma isolates were positive for PCR, showing a band of 574-bp (Figure 3). All the PCR positive samples were characterized as M. mycoides subsp. mycoides by PCR-RFLP method (Figure 4). Discussion The isolation rate of 6.87% of M.  mycoides subsp.  mycoides found in this study was slightly higher than the previous investigations carried out in Northern and Central Nigeria (Ankeli et al. 2016, Musa et al. 2016, Tambuwal and Egwu 2017). The increased rate observed in the same agro-ecological zone may be connected to the social security situation in the North-eastern Nigeria, where there are fragmented veterinary services due to insurgency (Aliyu species were shipped to the above mentioned WOAH Reference Laboratory for CBPP, where 37  isolates were confirmed on both PPLO media and PCR specific for Mycoplasma species. Thus, 19 (10.56%) lung tissues, 17 (9.44%) pleural fluid and 1 (1.67%) ear swab yielded positive results, leading to an overall Mycoplasma species recovery rate of 7.70% (37/480), out of which Adamawa State had 25 (8.80%) and Taraba States had 12 (6.12%). More isolates came from Adamawa State compared to Taraba State. The isolation and recovery rates of Mycoplasma species are shown on Table I. Mycoplasma mycoides subsp. mycoides (Figure 2) was the dominant species 33 (6.87%). In Adamawa State, 12 (10.91%) isolates of M. mycoides subsp. mycoides came from both, lung tissues and pleural fluids. The study revealed that in Taraba State 5  (7.14%) and 4  (5.71%) isolates of M.  mycoides subsp.  mycoides came from lung tissues and pleural fluid, respectively. The samples from nasal and ear swab from the study Table I. Isolation rates of Mycoplasma species and identification of M. mycoides subsp. mycoides (Mmm) from cattle in Adamawa and Taraba States, Nigeria. Source of sample Sample type No. of samples collected No. of Mycoplasma cultured (%) No. of Mycoplasma confirmed (%) No. of Mmm identified (%) Adamawa State Lungs 110 12 (10.91) 12 (10.91) 12 (10.91) Pleural fluid 110 13 (11.82) 13 (11.82) 12 (10.91) Nasal swab 32 0 (0.00) 0 (0.00) 0 (0.00) Ear swab 32 0 (0.00) 0 (0.00) 0 (0.00) Sub-total 284 25 (8.80) 25 (8.80) 24 (8.45) Taraba State Lungs 70 8 (11.43) 7 (10.00) 5 (7.14) Pleural fluid 70 5 (7.14) 4 (5.71) 4 (5.71) Nasal swab 28 0 (0.00) 0 (0.00) 0 (0.00) Ear swab 28 1 (3.57) 1 (3.57) 0 (0.00) Sub-total 196 14 (7.14) 12 (6.12) 9 (4.59) TOTAL 480 39 (8.13) 37 (7.71) 33 (6.87) Figure 2. Colonies of M. mycoides subspecies mycoides from lung sample on PPLO agar showing characteristic ‘fried egg shape’ colonies, dense raised central area ‘nipple’ (n) and transparent periphery (t) growth on PPLO agar isolated at NVRI Vom after 72 hours (A) and recovered from isolates shipped to IZSAM Teramo, Italy after 5 days (B) of incubation viewed under stereomicrospe (X35). A B Fried egg colony with dark centre n t 291Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 Francis et al. M. mycoides subsp. mycoides in Nigeria fragments delineated M.  mycoides subsp.  mycoides from M.  mycoides subspecies capri. This finding corroborates the earlier reports (Bashiruddin et  al. 1999, Musa et  al. 2016) and it further revealed strains of M. mycoides subsp. mycoides circulating in Adamawa and Taraba States, Nigeria. In conclusion, the present study has established the occurrence of M.  mycoides subsp.  mycoides North-eastern part of Nigeria. Also, a high number of M. mycoides subsp. mycoides field strains have been isolated which is to the best of our knowledge the first figure of this kind in Nigeria. It is recommended to strenghten animal movements control at Nigerian national and international borders, in order to minimise the spread of this dreaded and internationally recognised disease of cattle. Statement of animal rights As at the time of kick-starting this study, Ahmadu Bello University Zaria, Nigeria did not have any committee mandated to authorise research works. Though, the research procedure employed in this study was presented to the Postgraduate Board of Faculty of Veterinary Medicine, which considered ethical and welfare standards before approval was given. Acknowledgements The authors appreciate the staff of Mycoplasma Laboratory, National Veterinary Research Institute Vom, Plateau State Nigeria and the management of WOAH Reference Laboratory for CBPP at Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise ‘G Caporale’, Campo Boario Teramo, Italy for free bench space and technical support for this work. et  al. 2000, Francis et  al. 2018a). To the best of our knowledge, this is the highest reported isolation rate of M.  mycoides subsp.  mycoides from field samples in Nigeria. The high isolation rate of M.  mycoides subsp. mycoides obtained in this study may indicate that the disease is very active and endemic in the study area. The increased and unrestricted cattle movements, as well as the porous nature of borders with neighbouring countries, might have among other factors contribute to the high rate observed in this study (Aliyu et al. 2000). Mycoplasma mycoides subspecies  mycoides was not isolated from the nasal and ear swab samples collected, even though WOAH recommended these sites for sampling in live animals (Karahan et  al. 2010, OIE 2015). This may be due to contamination of such sites with debris, ear mites, other bacterial and Mycoplasma flora, thus suppressing M. mycoides subsp.  mycoides from thriving in such anatomical sites (Santos et al. 2009). Compared to nasal and ear swabs, the broncho-alveolar lavage (BAL) method of sampling is the most preferred method for isolation of Mycoplasma in live animals, to avoid sample contamination. However, the BAL sampling method was much more difficult to perform under field conditions, and animal owners are usually reluctant to allow this procedure (Karahan et  al. 2010, Akan et al. 2014). The results obtained in the present study are in contrast with the previous report of Ankeli and colleagues (Ankeli et  al. 2016), who reported isolation of M. mycoides subsp. mycoides from the ear canal of apparently healthy cattle in Plateau State. Molecular typing of thirty-three isolates out of the original thirty-seven was quite significant and showed that CBPP caused by M.  mycoides subsp. mycoides was active, endemic and widespread in the study area. This finding is in agreement with previous reports (Bashiruddin et  al. 2005, Muuka et  al. 2013, Wade et  al. 2015, Musa et  al. 2016). The digested restriction endonuclease 380-bp Figure 3. PCR amplification of M. mycoides subspecies CAP‑21 genomic region (574‑bp) on 1% agarose gel for identification using primers MM450 and MM451. Lane M = Molecular size marker 100-bp; Lanes 1-4 = Samples showing Mm subspecies; PC = Positive control; NC = Negative control. Figure 4. PCR‑RFLP amplicons on 3% agarose gel for the identification of M. mycoides subspecies mycoides following digestion of PCR product with restriction endonuclease Vsp1. Lane M = Molecular size marker 100-bp; Lanes 1-5 = Positive samples showing M. mycoides subsp. mycoides profile; lane PC1 = M. mycoides subsp. mycoides positive control; lane PC2 = Mm subsp. capri positive control. M 1 2 3 4 PC NC M 600 500 400 574 bp M 1 2 3 4 PC1 PC2 M5 400 300 150 380 bp 230 bp 180 bp 150 bp 292 Veterinaria Italiana 2022, 58 (3), 287-293. doi: 10.12834/VetIt.2331.16051.3 M. mycoides subsp. mycoides in Nigeria Francis et al. Aliyu M.M., Obi T.U. & Egwu G.O. 2000. Prevalence of contagious bovine pleuropneumonia (CBPP) in northern Nigeria. Prev Vet Med, 47, 263-269. Akan M., Babacan O., Torun E., Mustak H.K. & Oncel T. 2014. Diagnosis of Mycoplasma bovis infection in cattle by ELISA and PCR. Kafkas University Vet J, 20, 249-252. Ameh J.A., Nawathe D.R. & Emmans T.A. 1998. 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