The Predominant Incidence of Mycoplasma mycoides subsp. capri in Suspected Cases of Contagious Caprine Pleuropneumonia in Sheep and Goats of Northern Pakistan

The current study was approved by the ethical committee of the University of Agriculture Peshawar, and all experimental work including sample collection was carried out according to the national and institutional guidelines of animal ethics.

Samples were collected from two districts, District Buneer and Swat, of Khyber Pakhtunkhwa province. District Swat and Buneer are located in the North of Khyber Pakhunkhwa Province Pakistan. Nomads of these districts usually bring their animals down to the plain areas during winter (November to February) while return back to the hilly mountains for pastures during summer (March-October).

Samples were collected between October 2013 and April 2014. A total of 300 samples (the nasal discharge, pleural fluid and lung tissues) were collected at farms, herds and abattoirs from those suspected of CCPP, and were transferred to DifcoTM PPLO broth (Becton Dickinson, Sparks, MD, USA), and transported in ice box to our Mycoplasma reference laboratory at the Faculty of Animal and Veterinary Science, University of Agriculture, Peshawar.

All samples were streaked into PPLO media and incubated in anaerobic incubator (New Brunswick, Galaxy 48-S UK) with 5% CO2 at 37°C for 3-7 days for the isolation of mycoplasma species as described previously (Shahzad et al., 2012; OIE, 2014). The identification of the Mycoplasma was made by the appearance of typical fried egg or nipple live of 0.1 to 1 mm in diameter with a dense raised center in the middle.

Bacterial DNA was extracted using TIANamp Bacteria DNA Kit (TIANGEN, Neijing, China) according to the manufacturer’s instructions. The extracted DNA was then subjected to cluster specific PCR amplification with a set of primer MmF 5’-CGA AAG CGG CTT ACT GGC TTG TT-3’ and MmR 5’-TTG AGA TTA GCT CCC CTT CAC AG-3’, followed by specie specific PCR for Mccp with primer set Mccp.spe-F: 5’-ATCATTTTTAATCCCTTCAAG-3’, and Mccp.spe-R: 5’-TACTATGAGTAATTATAATATATGCAA-3 and Mmc P4-F: 5’-ACTGAGCAATTCCTCTT-3’ P6-R: 5’- TTAATAAGTCTCTATATGAAT-3’ was performed as described previously (Hotzel et al., 1996; Manso-Silván et al., 2007). A PCR product of 548 bp for MM cluster, 194 bp for Mmc and 316 bp for Mccp was considered positive.

Mccp, Mycoplasma capricolum subsp. Capripneumoniae; Mmc, Mycoplasma mycoides subsp. capri.

Of 300 samples, 79 (26%) resulted mycoplasma like growth. A total of 100 samples were collected from nasal swab, and 25 (25%) were found positive for mycoplasma growth containing 10/50 samples (20%) from sheep, while 15/50 (30%) were from goats (See Supplementary Fig. S1). Similarly, a total of 100 samples (50 from sheep and 50 from goats) from pleural fluid with 33% indicated positive growth such as 15/50 (30%) were found positive from sheep and 18/50 (36%) from goats. Finally, of the 100 samples obtained from lung tissue, a total of 21 were found positive such as 9/50 (18%) were found positive for growth of mycoplasma from sheep, while 12/50 (24%) were derived from goats. Interestingly, 28% of samples from District Buneer were cultured positive, while 33% from District Swat. Overall, our study shows that pleural fluid is relatively preferred for mycoplasma isolation from suspected cases.

Results of the MM cluster specific PCR revealed a total of 49 (16.33%) samples were positive (Table I). All samples that were found positive for MM cluster PCR were then subjected to specie specific PCR for the identification of Mmc and Mccp (not shown). Results indicated that only 15 (5%) samples were found positive for Mccp, while 34 (11.3%) samples were PCR amplified against Mmc. Interestingly, three of the samples were found positive both for Mmc and Mccp suggesting mixed infection. Overall, the isolation of both Mmc and Mccp from suspected cases of CCPP suggests their involvement in the development of the diseases.

Mycoplasmosis is an important respiratory disease of small ruminants, causing heavy economic losses throughout the country (Sadique et al., 2012; Abbas et al., 2018). Members of the Mycoplasma mycoides could lead to a severe respiratory syndrome called as CCPP. Mccp is the principal cause of CCCP in small ruminants; however, there are reports that other members of MM cluster may potentially cause CCPP. There is often outbreak of CCPP in small ruminants in the Northern region of Pakistan; however, the nature of the etiological agent confirmed by PCR has never been reported. The current study was thus aimed to identify members of MM involve in the development of CCPP.

Although, this is established that the principal cause of CCPP is known to be Mccp, however in Asia and Africa, isolation of Mmc from suspected cases of CCPP suggests its involvement and an unknown role in pathogenesis. Our first report on the isolation of Mmc as an etiological agent of CCPP in goats in central District of Peshawar in Khyber Pakhtunkhwa province prompted us to investigate further the prevalence of Mmc in suspected cases of CCPP mainly in the Northern region of Pakistan (Shah et al., 2017). Notably, in the current study Mmc was found significantly highly prevalent (11%) as compared to Mccp (5%). Furthermore, quite surprisingly, the incidence rate of Mccp was found significantly lower (5%) in the Northern regions as compared to our previous report of 24% in the central district of Peshawar-Khyber Pakhtunkhwa. Other reports from Pakistan indicate isolation and identification of Mycoplasma mycoides subspecies Capri (Awan et al., 2009), Mycoplasma capricolum subspecies capricolum (Awan et al., 2010; Shah et al., 2017), Mycoplasma putrefaciens (Awan et al., 2009) and Mycoplasma capricolum subspecies capripneumoniae (Awan et al., 2009; Shahzad et al., 2013).

In the present study Mycoplasma was successfully grown on modified PPLO media for isolation from samples originated from different sources of suspected animals including nasal discharges, pleural fluid and lung tissues. It is observed that better isolation was achieved from the pleural fluid of necropsied and sacrificed animals. These findings are in full agreement with previous studies (Thiaucourt et al., 2000; Sadique et al., 2012). Of the 49, only 15 were confirmed for Mycoplasma capricolum subspecies capripneumoniae through PCR from different sources such as nasal discharges 6 (12.24%), pleural fluid 4 (8.16 %) and lung tissues, 5 (10.20 %) respectively. While 34 out of 49 positive were confirmed as Mycoplasma mycoides subspecies Capri from different sources of samples such as 13 (26.53 %) from nasal discharges, 14 (28.57 %) from pleural fluid and 7 (14.28 %) from lung tissues, respectively. Possibly, this is more likely due to the fact that Mycoplasma is intracellular microorganism surrounded by cell membrane containing lypoglycan. This initiates the acute inflammatory response in host with severe exudation. The survival percentage of the Mycoplasma increases when it is present in the deep tissues of the host by evading immune system. Antimicrobial resistance is swiftly increasing all over the world including Pakistan (Ali et al., 2016, 2017; Khattak et al., 2018) and our current report suggested that mycoplasma species isolated from suspected cases of CCPP have been found resistant to tylosin, a favorite drug used against the condition (Shah et al., 2017), confirms this notion. Identification of the prevalent isolates could thus be used for determining susceptibility profile in order to avoid empirical treatment and emergence of antibiotic resistance. In conclusion, our study indicates that Mmc could be actively involved in the development of CCPP infections in sheep and goats in Pakistan, and thus an overall epidemiological survey of the Northern region should be initiated in the country.

The current study concluded a high prevalence of Mycoplasma mycoides subspecies capri followed by Mycoplasma capricolum subspecies capripneumoniae in the suspected cases of CCPP from sheep and goats of Northern region of Pakistan. The higher isolation rate of Mycoplasma mycoides subspecies Capri suggests its involvement in the disease, and a proper and comprehensive survey should be carried out in order to understand the frequency of members of MM cluster involved in the development of CCPP infection in Pakistan.

The authors are grateful to the Pakistan Science Foundation for providing financial assistance to conduct this study. This study was carried out under the PSF project No. PSF/NSLP/KP-AU (219). We are also thankful to Dr. Waseem Shahzad, Veterinary Research Institute, Lahore, Pakistan for his technical assistance in the isolation and confirmation of local isolates, and Dr. F. Thiaucourt and Manso Silvan from CIRAD, France for their excellent technical assistance and providing positive controls of Members of MM cluster.

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2018.50.5.sc9

Authors have declared no conflict of interest.