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Isolation rate and antimicrobial susceptibility profiles of Mycoplasma mycoides subspecies capri field isolates from sheep and goats in Pakistan
Small Ruminant Research 153 (2017) 118–122
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Isolation rate and antimicrobial susceptibility profiles of Mycoplasma mycoides subspecies capri field isolates from sheep and goats in Pakistan
MARK
Muhammad Kamal Shaha, Umer Saddiquea, Shakoor Ahmada, Yousaf Hayatb, ⁎ Sadeeq ur Rahmanc, Mohammad Farooque Hassand, Tariq Alid, a
Department of Animal Health, The University of Agriculture, Peshawar, Pakistan Department of State, Mathematics and Computer Sciences,The University of Agriculture, Peshawar, Pakistan c College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan d College of Veterinary Medicine, China Agricultural University, Beijing, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycoplasma mycoides subsp. capri Mycoplasmosis Antimicrobial resistance Sheep and goats
Mycoplasmosis, the respiratory infection, is a well-established cause of heavy economic losses in sheep and goats farming. The occurrence and antibiotics susceptibility profiles of clinical isolates of Mycoplasma mycoides subsp. capri (Mmc) from suspected cases of contagious caprine pleuropneumonia (CCPP) were investigated in small ruminants of three climatic- southern, central and northern- regions of Khyber Pakhtunkhwa province of Pakistan. Specie specific-PCR for Mmc confirmed 24% (198/825) isolates with significantly (P ˂ 0.001) higher number (83/275; 30.18%) recovered from the northern-, followed by southern- (68/275; 24.73%) and centralregions (47/275; 17.09%), respectively. Subsequently, a total of 54 PCR-confirmed isolates (18 from each zone) were subjected to antibiogram assay. Five different common antimicrobial agents including ceftiofur, enrofloxacin, gentamycin, oxytetracycline and tylosin were tested by disc diffusion and broth microdilution methods The antibiotic susceptibility profiles of Mmc isolates indicated that enrofloxacin exhibited the highest (P ˂ 0.001) in vitro antibacterial activity with a maximum zone of inhibition (19 ± 0.71 mm) and the lowest MIC (0.001 ± 0.0002 mg/mL) amongst the tested antimicrobial agents, followed by gentamycin (11 ± 0.45 mm and 0.01 ± 0.001 mg/mL zone of inhibition and MIC, respectively). All these isolates were found resistant against tylosin, oxytetracycline and ceftiofur sodium: antibiotics that are favored in clinical cases of CCPP. This study concludes a high occurrence of Mmc in small ruminants during infection of CCPP, and increased resistance rate against commonly used antibiotics.
1. Introduction Ruminant mycoplasmosis, an important respiratory tract infection considered to cause heavy economic losses, is often reported from countries of Africa and Asia including Pakistan (Sadique et al., 2012; Shahzad et al., 2013). Pakistan is ranked 3rd in goat- and 12th in sheepproduction and sheep and goat sector contributes considerably in the overall economy of the country (Economic Survey, 2013). Mycoplasmosis, a principal cause of acute respiratory syndrome that usually terminated in chronic complications, lead to huge economic losses in the form of diminished production, treatment cost, high mortality and decreased export, and is considered as serious threat for the productivity of sheep and goats (Nicholas et al., 2008; OIE, 2017). Contagious caprine pleuropneumonia (CCPP), a respiratory disease syndrome, is caused by a group of six closely related microorganisms known as Mycoplasma mycoides (Mm) cluster. Mycoplasma mycoides ⁎
subsp. capri (Mmc) is one of the most prevalent pathogenic species among the Mm cluster (Manso-Silvan et al., 2007). Mmc could cause CCPP in addition to complications like arthritis, mastitis, keratoconjunctivitis (altogether known as contagious agalactia), urogenital infection and occasionally causing abortion in the pregnant ewes (Mondal et al., 2004; Nicholas et al., 2008). CCPP commonly occurs in septicemic form accompanied by high morbidity and mortality (Sadique et al., 2012), and is listed in the OIE-listed diseases, infection and infestations 2017 (OIE, 2017). Although, CCPP is generally caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), which affects thoracic cavity mainly (OIE, 2017); however, in Asia and Africa, Mmc has been reported frequently associated with CCPP (Gelagay et al., 2007; Mondal et at., 2004; Sadique et al., 2012; Shahzad et al., 2012). The disease is contagious in nature, which rapidly spreads among animals by direct contact through oculonasal discharge, excretion from wound, open joints, urine and faeces (OIE, 2017).
Corresponding author at: College of Veterinary Medicine, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, 100193, Beijing, PR China. E-mail addresses: [email protected], [email protected] (T. Ali).
http://dx.doi.org/10.1016/j.smallrumres.2017.06.002 Received 20 February 2017; Received in revised form 23 May 2017; Accepted 1 June 2017 Available online 03 June 2017 0921-4488/ © 2017 Elsevier B.V. All rights reserved.
Small Ruminant Research 153 (2017) 118–122
M.K. Shah et al.
Fig. 1. Map of Khyber Pakhtunkhwa province of Pakistan, showing the three regions i.e. northern, central and southern, which were sampled for the detection of Mycoplasma mycoides subsp. capri.
2. Material and methods
Table 1 Isolation rate of Mycoplasma mycoides subsp. capri in suspected of contagious caprine pleuropneumonia in small ruminants. Climatic Zones
Northern Central Southern Total
Number % within Number % within Number % within Number % within
Status on PCR
zone zone zone zone
Positive
Negative
83 30.18 47 17.09 68 24.73 198 24.0
192 69.81 228 82.91 207 75.27 627 76.0
Total
275 100.0 275 100.0 275 100.0 825 100.0
Chi-square
13.03
2.1. Sampling, isolation and identification of Mycoplasma mycoides subsp. capri (Mmc)
P-value
The clinical Mmc isolates were recovered from sheep and goats suffering from respiratory complications from three different climatic regions viz., northern, central and southern of Khyber Pakhtunkhwa (KPK), Pakistan during December 2014 to May 2016 (Fig. 1). A total of 825 clinical samples, including nasal (n = 450), tracheal swabs (n = 300) and lungs tissue (n = 75) were collected from animals suspected of contagious caprine pleuropneumonia (CCPP). Of these samples, 412 and 413 were collected from sheep and goats, respectively. From each zone, 275 samples, including nasal = 150, tracheal = 100 and lungs = 25, were collected. Samples were collected with sterile swabs, transferred into Difco™ PPLO broth (Becton Dickinson, Sparks, MD), and transported in ice box to our Mycoplasma reference laboratory, Faculty of Animal and Veterinary Science, University of Agriculture, Peshawar. All collected samples were incubated in anaerobic incubator (New Brunswick, Galaxy 48-S, UK) with 5% CO2 at 37 °C for 3–7 days. The incubated test tubes were examined daily for presence of mass turbidity, whirling movement and change in color. The positive growth sample was sub-cultured on Difco™ PPLO agar for the appearance of nipple like or fried egg shape typical mycoplasma colonies. The desired colonies were sub-cultured three times to obtain pure culture
0.001
CCPP is usually treated with antibiotics like enrofloxacin, gentamycin, kanamycin, oxytetracycline, tylosin and third generation cephalosporins such as ceftiofur. However, due to indiscriminate use of these antimicrobials drug resistance have been recently developed with cases of therapeutic failure (Citti and Blanchard, 2013; Scott and Menzies, 2011). Therefore, the current study was carried out with objectives to investigate the occurrence and antimicrobial susceptibility profiles of clinical isolates of Mmc from diverse localities of Khyber Pakhtunkhwa.
119
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Fig. 2. Isolation of Mycoplasma mycoides subsp. capri. A. Isolation rate from different clinical samples, i.e. nasal discharge, tracheal swab & lungs tissue of animals. B. Isolation rate from sheep and goats in three climatic zones of Khyber Pakhtunkhwa, Pakistan. Y-axis shows the incidence rate (%) of Mmc isolates; X-axis indicates the isolation from three climatic zones.
test and one way analysis of variance (ANOVA) was used to check the statistical difference between different treatments used for antimicrobial assay. P value of ≤ 0.05 was considered as significant.
according to the standard protocol of the Office International des Epizootics (OIE, 2017). 2.2. Confirmation of Mycoplasma mycoides subsp. capri
3. Results For the confirmation of Mycoplasma spp., the pure culture was subjected to DNA extraction by TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The species specific primers P4-F (5′-ACT GAG CAA TTC CTC TT-3′), P6-R (5′TTA AAT AAG TTT GTA TAT GAA T-3′) were used for the molecular confirmation of Mmc as previously described by (Hotzel et al., 1996).
Out of the total 825 suspected cultured samples, 271 (32.8%) showed positive growth of mycoplasma i.e. turbidity and whirling movement in PPLO broth. Subsequently, these positively cultured samples were subcultured onto PPLO agar media, and 233 (28.2%) samples produced typical nipple like colonies suggesting Mycoplasma spp. Then, 198 (24%) were confirmed as Mycoplasma mycoides subsp. capri (Mmc) by using species specific PCR assay. PCR results revealed that 83/275 (30.18%), 68/275 (24.73%) and 47/275 (19.09%) of Mmc isolates were confirmed from northern, southern and central zones, respectively (Table 1). The occurrence of Mmc was found significantly (P ˂ 0.001) higher in northern zone. The highest numbers of clinical isolates were recovered from lungs tissues, followed by nasal discharge. The distribution of recovered isolates from different clinical samples like nasal discharge, tracheal swab and lung tissues are presented in Fig. 2A. The isolation rate of Mmc was significantly higher in goats as compared with sheep in all three climatic zones (Fig. 2B). The results of antibiotic susceptibility testing showed that enrofolaxacin has the maximum activity against Mmc isolates with the largest zone of inhibition (19 ± 0.71 mm), followed by gentamycin (11 ± 0.45 mm). Mmc isolates displayed lower susceptibility to tylosin (6.8 ± 0.37 mm), but were totally resistant to oxytetracycline (3 ± 0.32 mm) and ceftiofur sodium (0 ± 0.0 mm). Similar, results were also observed by MIC method, enrofolaxacin displayed the lowest MIC value (0.001 mg/mL) against the Mmc isolates, followed by gentamycin (MIC = 0.01 mg/mL). The other tested antimicrobial agents such as tylosin, oxytetracycline and ceftiofur sodium showed MIC values of 01, 10 and 10 mg/mL, respectively (Fig. 3).
2.3. Antimicrobial susceptibility of clinical isolates To evaluate in vitro antimicrobial activity, five common antimicrobial agents were tested against confirmed 54 clinical Mmc isolates by randomly choosing eighteen isolates from each region. The standard disc diffusion and the minimum inhibition concentrations (MICs), by micro broth dilution, methods were adopted for determination of antibiotic susceptibility following guide lines of the Clinical and Laboratory Standard Institute and Hannan (Hannan, 2000; CLSI, 2002). The antimicrobial discs (Becton Dickinson, Sparks, MD, USA) including ceftiofur sodium (10 μg), enrofloxacin (10 μg), gentamycin (10 μg), oxytetracycline (10 μg) and tylosin (10 μg), which are commonly used antibiotics for pulmonary infection in ruminants. A 10 μL of Mycoplasma culture containing approximately 1 × 107–1 × 108 CFU/mL was streaked onto PPLO agar plates. Antibiotic disc was placed gently on agar plate at recommended distance and plates were incubated in 5% CO2 at 37 °C for 24–48 h. The results were interpreted in mm according to CLSI (CLSI, 2002). For comparison, negative control disc containing DMSO (Dimethyl sulfoxide; 10%) without any antimicrobial agent was included in the study. The MICs of above mentioned antimicrobials were also determined in 96 well microtitration plates (Corning, Corning, NY, USA) as described previously (Hannan, 2000). Antimicrobials were added to the microtitration plate in triplicate to make a final concentration of 10 mg/mL in the first wells and then serially diluted by 10 folds to make further concentrations of 1, 0.1, 0.01, 0.001, 0.0001 mg/mL of each tested drug. Ten μL of the PBS washed bacterial cell (OD600 = 0.3) was pipetted to each well of the microtitration plate except the negative control which contained PPLO broth. Mycoplasma putrefaciens, obtained from Veterinary Research Institute, Lahore, was used as a positive control strain.
4. Discussion In Pakistan, caprine mycoplasmosis, which is mainly caused by important pathogenic species of Mycoplasma mycoides subsp. Capri (Mmc), is a serious threat to small ruminant production (Banaras et al., 2016; Waseem et al., 2013). The present study reported a significantly higher occurrence of Mmc in the northern zone of KPK. The possible reason of higher isolation rate from this zone might be the intense cold climatic conditions that cause stress and predispose the animals to immunosuppression and consequently make it susceptible to respiratory complications. The Mmc isolates were successfully isolated and confirmed from different clinical samples like nasal discharge, tracheal swab and lung tissues. Isolation of Mmc has also been
2.4. Statistical analysis Chi- Square test (χ2) was used for testing the association among the selected zones and sources of samples using SPSS v.19.0. The student t120
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Fig. 3. Comparative antimicrobial susceptibility of clinical Mycoplasma mycoides subsp. capri isolates against five common antibiotics using micro broth dilution method.
aminoglycosides, fluoroquinolones, tetracyclines and marcolides are common agents used in animals against mycoplasmosis (Antunes et al., 2007). Nevertheless, the microbial resistance against the common antibiotics is an emergent issue, which poses a serious health concern throughout the world (Ali et al., 2016). At the dawn of discovery of these antimicrobial agents most pathogenic organisms were highly susceptible; however, the efficacy of these antibiotics has been decreased due to development of resistance (Gautier et al., 2002). The finding of the study revealed that enrofloxacin was the most potent therapeutic agent. These results were in accordance with the findings
previously reported from tracheal and nasal discharges (Banaras et al., 2016). The highest numbers of clinical isolates were recovered from goats as compared to sheep in the current study. This may be due to the reason that goats are more sensitive to different bacterial infections including mycoplasmosis. These findings are justified by a study in which 35% and 65% of Mycoplasma spp. were isolated from sheep and goats, respectively (Al-Momani et al., 2006). Mycoplasmosis is mainly treated with different antimicrobials with various degree of success, as vaccination has not been an effective tool against this disease (Citti and Blanchard, 2013). Antimicrobials like 121
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ESBL-producing Escherichia coli from cows suffering mastitis in china contain clinical class 1 integrons with CTX-M linked to ISCR1. Front. Microbiol. http://dx.doi.org/10. 3389/fmicb.2016.01931. Antunes, N.T., Tavío, M.M., Assunção, P., Rosales, R.S., Aquili, V., De la Fé, C., Poveda, J.B., 2007. In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials. Vet. Microb. 119, 72–75. Ayling, R.D., Bisgaard-Frantzen, S., March, J.B., Godinho, K., Nicholas, R.A.J., 2005. Assessing the in vitro effectiveness of antimicrobials against Mycoplasma mycoides subsp. mycoides small-colony type to reduce contagious bovine pleuropneumonia infection. Antimicrob. Agents Chemother. 49, 5162–5165. Banaras, F., Abbas, F., Awan, M.A., Riaz, A., Khan, I.A., Mengal, M.A., Banaras, S., Murtaz-ul-Hassan Zafar, M.A., Baig, R.M., 2016. Molecular survey on the prevalence of Caprine mycoplasma in the goats of Quetta City of Pakistan. Inter. J. Vet. Sci. 5, 158–163. CLSI, 2002. Clinical and Laboratory Standard Institute, development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. CLSI document, approved guideline, 22 Wayne, Pennsylvania, USA. Citti, C., Blanchard, A., 2013. Mycoplasmas and their host: emerging and re-emerging minimal pathogens. Trends Microbiol. 21, 196–203. Economic Survey, 2013. Govt. of Pakistan an, Finance Division, Economic Advisors Wing, Ministry of Finance Islamabad, Pakistan. Gautier, A.K., Kobisch, Reinhardt, Kempf, M., 2002. In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae. Vet. Microbiol. 88, 47–58. Gelagay, A., Teshale, S., Amsaluc, W., Esayas, G., 2007. Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia. Small Rumin. Res. 70, 131–135. Hannan, P.C., 2000. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary Mycoplasma species. Vet. Res. 31, 373–395. Hotzel, H., Sachse, K., Pfützner, H., 1996. A PCR scheme for differentiation of organisms belonging to the M. mycoides cluster. Vet. Microbiol. 49, 31–43. Loria, G.R., Sammartino, R.A., Nicholas, R.D., 2003. In vitro susceptibilities of field isolates of Mycoplasma agalactiae to oxytetracycline, tylosin, enrofloxacin, spiramycin and lincomycin-spectinomycin. Res. Vet. Sci. 75, 3–7. Manso-Silvan, L., Perrier, X., Thiaucourt, F., 2007. Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group. Int. J. Sys. Evo. Microbiol. 57, 2247–2258. Mondal, D., Pramanik, A.K., Basak, D.K., 2004. Clinico-Haematology and pathology of Caprine Mycoplasma pneumonia in rain fed tropics of West Bengal. Small Rumin. Res. 51, 285–295. Nicholas, R., Ayling, R., McAuliffe, L., 2008. Respiratory diseases of small ruminants. In: Nicholas, R., Ayling, R., McAuliffe, L. (Eds.), Mycoplasma Diseases of Ruminants. CABI, Wallingford, UK, pp. 171–179. OIE, 2017. OIE-listed Diseases, Infections and Infestations in Forcein 2017. Office International DesEpizootics, World Organization for Animal Health, Paris, France (Accessed 20 May 2017). http://www.oie.int. Sadique, U., Chaudhry, Z.I., Younas, M.R., Anjum, A.A., Hassan, Z., Sajid, A., Mushtaq, M., 2012. Pathogenesis and immunohistochemical studies of caprine pleuropneumonia in experimentally infected goats. Pak Vet. J. 32, 427–731. Scott, L.C., Menzies, P.I., 2011. Antimicrobial resistance and small ruminants veterinary practice. Vet. Clin. North Am. Food Anim. Pract. 1, 23–32. Shahzad, A., Rashid, M., Muhammad, S.K., Mansur, D.A., Mohammad, I., Rashid, A., 2012. Characterization, molecular diagnosis and prevalence of caprine mycoplasmosis in different areas of Pakistan. Pak. J. Zool. 44 (2), 559–568. Waseem, S., Rashid, M., Mohammad, R., Rashid, A., Mohammad, S.K., Ghulam, A., Mohammad, I., Fayyaz, M., 2013. Prevalence, molecular diagnosis and treatment of Mycoplasma conjunctivae isolated from infectious keratoconjunctivitis affected Lohi sheep maintained at Livestock Experiment StationBahadur Nagar,Okara, Pakistan. Trop. Anim. Health Prod. 45, 737–742.
that enrofloxacin was considered as the most efficacious among the antimicrobial agents against mycoplasma infection (Hannan, 2000). Similar findings are also reported by Loria et al. (2003), they screened different antibiotics by MIC method against 24 isolates of M. agalactiae and found that enrofloxacin was the most effective agent with lowest MIC of 0.25 μg/mL. The study of Antunes et al. (2007 Antunes) also revealed that enrofloxacin and its metabolites were effective against some species of mycoides cluster like M. mycoides subspp. large colony and M. capricolum subspp. capricolum. Generally, marcolides such as tylosin is considered as the drug of choice and is extensively used in practice for the treatment of caprine, bovine and avian mycoplasmosis. However, Mycoplasma spp. has great potential to develop resistance against antimicrobial agents due to rapid mutational changes. The striking findings of this study revealed that all the local isolates were found resistant against tylosin. This could reflect indiscriminate use of tylosin. Oxytetracycline and ceftiofur sodium produced higher MIC values and lower zones of inhibition indicating intermediate resistance. In several countries of Europe, antimicrobial resistance of Mycoplasma spp. has been reported against tylosin, oxytetracycline and spectinomycin (Ayling et al., 2005). To the best of our knowledge, this is the first time comprehensive study the occurrence of clinical isolates of Mmc from diverse localities of Khyber Pakhtunkhwa. 5. Conclusions The present study concluded high occurrence of Mmc associated with cases of CCPP in small ruminants of Khyber Pakhtunkhwa province. In addition, we observed an increased resistance against commonly used antibiotics such as tylosin. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to the Pakistan Science Foundation (PSF) for providing financial assistance to conduct this study. We are also thankful to Dr. Waseem Shahzad, Veterinary Research Institute, Lahore for his technical assistance in the isolation and confirmation of local isolates. References Al-Momani, W., Halablab, M.A., Abo-Shehada, M., Katie, M., McAuliffe, L., Nicholas, R.A.J., 2006. Isolation and molecular identification of small ruminant mycoplasmas in Jordan. Small Rumin. Res. 65, 106–112. Ali, T., ur Rahman, S., Zhang, L., Shahid, M., Zhang, S., Liu, G., Gao, J., Han, B., 2016.
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Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Isolation rate and antimicrobial susceptibility profiles of Mycoplasma mycoides subspecies capri field isolates from sheep and goats in Pakistan
MARK
Muhammad Kamal Shaha, Umer Saddiquea, Shakoor Ahmada, Yousaf Hayatb, ⁎ Sadeeq ur Rahmanc, Mohammad Farooque Hassand, Tariq Alid, a
Department of Animal Health, The University of Agriculture, Peshawar, Pakistan Department of State, Mathematics and Computer Sciences,The University of Agriculture, Peshawar, Pakistan c College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan d College of Veterinary Medicine, China Agricultural University, Beijing, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycoplasma mycoides subsp. capri Mycoplasmosis Antimicrobial resistance Sheep and goats
Mycoplasmosis, the respiratory infection, is a well-established cause of heavy economic losses in sheep and goats farming. The occurrence and antibiotics susceptibility profiles of clinical isolates of Mycoplasma mycoides subsp. capri (Mmc) from suspected cases of contagious caprine pleuropneumonia (CCPP) were investigated in small ruminants of three climatic- southern, central and northern- regions of Khyber Pakhtunkhwa province of Pakistan. Specie specific-PCR for Mmc confirmed 24% (198/825) isolates with significantly (P ˂ 0.001) higher number (83/275; 30.18%) recovered from the northern-, followed by southern- (68/275; 24.73%) and centralregions (47/275; 17.09%), respectively. Subsequently, a total of 54 PCR-confirmed isolates (18 from each zone) were subjected to antibiogram assay. Five different common antimicrobial agents including ceftiofur, enrofloxacin, gentamycin, oxytetracycline and tylosin were tested by disc diffusion and broth microdilution methods The antibiotic susceptibility profiles of Mmc isolates indicated that enrofloxacin exhibited the highest (P ˂ 0.001) in vitro antibacterial activity with a maximum zone of inhibition (19 ± 0.71 mm) and the lowest MIC (0.001 ± 0.0002 mg/mL) amongst the tested antimicrobial agents, followed by gentamycin (11 ± 0.45 mm and 0.01 ± 0.001 mg/mL zone of inhibition and MIC, respectively). All these isolates were found resistant against tylosin, oxytetracycline and ceftiofur sodium: antibiotics that are favored in clinical cases of CCPP. This study concludes a high occurrence of Mmc in small ruminants during infection of CCPP, and increased resistance rate against commonly used antibiotics.
1. Introduction Ruminant mycoplasmosis, an important respiratory tract infection considered to cause heavy economic losses, is often reported from countries of Africa and Asia including Pakistan (Sadique et al., 2012; Shahzad et al., 2013). Pakistan is ranked 3rd in goat- and 12th in sheepproduction and sheep and goat sector contributes considerably in the overall economy of the country (Economic Survey, 2013). Mycoplasmosis, a principal cause of acute respiratory syndrome that usually terminated in chronic complications, lead to huge economic losses in the form of diminished production, treatment cost, high mortality and decreased export, and is considered as serious threat for the productivity of sheep and goats (Nicholas et al., 2008; OIE, 2017). Contagious caprine pleuropneumonia (CCPP), a respiratory disease syndrome, is caused by a group of six closely related microorganisms known as Mycoplasma mycoides (Mm) cluster. Mycoplasma mycoides ⁎
subsp. capri (Mmc) is one of the most prevalent pathogenic species among the Mm cluster (Manso-Silvan et al., 2007). Mmc could cause CCPP in addition to complications like arthritis, mastitis, keratoconjunctivitis (altogether known as contagious agalactia), urogenital infection and occasionally causing abortion in the pregnant ewes (Mondal et al., 2004; Nicholas et al., 2008). CCPP commonly occurs in septicemic form accompanied by high morbidity and mortality (Sadique et al., 2012), and is listed in the OIE-listed diseases, infection and infestations 2017 (OIE, 2017). Although, CCPP is generally caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), which affects thoracic cavity mainly (OIE, 2017); however, in Asia and Africa, Mmc has been reported frequently associated with CCPP (Gelagay et al., 2007; Mondal et at., 2004; Sadique et al., 2012; Shahzad et al., 2012). The disease is contagious in nature, which rapidly spreads among animals by direct contact through oculonasal discharge, excretion from wound, open joints, urine and faeces (OIE, 2017).
Corresponding author at: College of Veterinary Medicine, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, 100193, Beijing, PR China. E-mail addresses: [email protected], [email protected] (T. Ali).
http://dx.doi.org/10.1016/j.smallrumres.2017.06.002 Received 20 February 2017; Received in revised form 23 May 2017; Accepted 1 June 2017 Available online 03 June 2017 0921-4488/ © 2017 Elsevier B.V. All rights reserved.
Small Ruminant Research 153 (2017) 118–122
M.K. Shah et al.
Fig. 1. Map of Khyber Pakhtunkhwa province of Pakistan, showing the three regions i.e. northern, central and southern, which were sampled for the detection of Mycoplasma mycoides subsp. capri.
2. Material and methods
Table 1 Isolation rate of Mycoplasma mycoides subsp. capri in suspected of contagious caprine pleuropneumonia in small ruminants. Climatic Zones
Northern Central Southern Total
Number % within Number % within Number % within Number % within
Status on PCR
zone zone zone zone
Positive
Negative
83 30.18 47 17.09 68 24.73 198 24.0
192 69.81 228 82.91 207 75.27 627 76.0
Total
275 100.0 275 100.0 275 100.0 825 100.0
Chi-square
13.03
2.1. Sampling, isolation and identification of Mycoplasma mycoides subsp. capri (Mmc)
P-value
The clinical Mmc isolates were recovered from sheep and goats suffering from respiratory complications from three different climatic regions viz., northern, central and southern of Khyber Pakhtunkhwa (KPK), Pakistan during December 2014 to May 2016 (Fig. 1). A total of 825 clinical samples, including nasal (n = 450), tracheal swabs (n = 300) and lungs tissue (n = 75) were collected from animals suspected of contagious caprine pleuropneumonia (CCPP). Of these samples, 412 and 413 were collected from sheep and goats, respectively. From each zone, 275 samples, including nasal = 150, tracheal = 100 and lungs = 25, were collected. Samples were collected with sterile swabs, transferred into Difco™ PPLO broth (Becton Dickinson, Sparks, MD), and transported in ice box to our Mycoplasma reference laboratory, Faculty of Animal and Veterinary Science, University of Agriculture, Peshawar. All collected samples were incubated in anaerobic incubator (New Brunswick, Galaxy 48-S, UK) with 5% CO2 at 37 °C for 3–7 days. The incubated test tubes were examined daily for presence of mass turbidity, whirling movement and change in color. The positive growth sample was sub-cultured on Difco™ PPLO agar for the appearance of nipple like or fried egg shape typical mycoplasma colonies. The desired colonies were sub-cultured three times to obtain pure culture
0.001
CCPP is usually treated with antibiotics like enrofloxacin, gentamycin, kanamycin, oxytetracycline, tylosin and third generation cephalosporins such as ceftiofur. However, due to indiscriminate use of these antimicrobials drug resistance have been recently developed with cases of therapeutic failure (Citti and Blanchard, 2013; Scott and Menzies, 2011). Therefore, the current study was carried out with objectives to investigate the occurrence and antimicrobial susceptibility profiles of clinical isolates of Mmc from diverse localities of Khyber Pakhtunkhwa.
119
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Fig. 2. Isolation of Mycoplasma mycoides subsp. capri. A. Isolation rate from different clinical samples, i.e. nasal discharge, tracheal swab & lungs tissue of animals. B. Isolation rate from sheep and goats in three climatic zones of Khyber Pakhtunkhwa, Pakistan. Y-axis shows the incidence rate (%) of Mmc isolates; X-axis indicates the isolation from three climatic zones.
test and one way analysis of variance (ANOVA) was used to check the statistical difference between different treatments used for antimicrobial assay. P value of ≤ 0.05 was considered as significant.
according to the standard protocol of the Office International des Epizootics (OIE, 2017). 2.2. Confirmation of Mycoplasma mycoides subsp. capri
3. Results For the confirmation of Mycoplasma spp., the pure culture was subjected to DNA extraction by TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The species specific primers P4-F (5′-ACT GAG CAA TTC CTC TT-3′), P6-R (5′TTA AAT AAG TTT GTA TAT GAA T-3′) were used for the molecular confirmation of Mmc as previously described by (Hotzel et al., 1996).
Out of the total 825 suspected cultured samples, 271 (32.8%) showed positive growth of mycoplasma i.e. turbidity and whirling movement in PPLO broth. Subsequently, these positively cultured samples were subcultured onto PPLO agar media, and 233 (28.2%) samples produced typical nipple like colonies suggesting Mycoplasma spp. Then, 198 (24%) were confirmed as Mycoplasma mycoides subsp. capri (Mmc) by using species specific PCR assay. PCR results revealed that 83/275 (30.18%), 68/275 (24.73%) and 47/275 (19.09%) of Mmc isolates were confirmed from northern, southern and central zones, respectively (Table 1). The occurrence of Mmc was found significantly (P ˂ 0.001) higher in northern zone. The highest numbers of clinical isolates were recovered from lungs tissues, followed by nasal discharge. The distribution of recovered isolates from different clinical samples like nasal discharge, tracheal swab and lung tissues are presented in Fig. 2A. The isolation rate of Mmc was significantly higher in goats as compared with sheep in all three climatic zones (Fig. 2B). The results of antibiotic susceptibility testing showed that enrofolaxacin has the maximum activity against Mmc isolates with the largest zone of inhibition (19 ± 0.71 mm), followed by gentamycin (11 ± 0.45 mm). Mmc isolates displayed lower susceptibility to tylosin (6.8 ± 0.37 mm), but were totally resistant to oxytetracycline (3 ± 0.32 mm) and ceftiofur sodium (0 ± 0.0 mm). Similar, results were also observed by MIC method, enrofolaxacin displayed the lowest MIC value (0.001 mg/mL) against the Mmc isolates, followed by gentamycin (MIC = 0.01 mg/mL). The other tested antimicrobial agents such as tylosin, oxytetracycline and ceftiofur sodium showed MIC values of 01, 10 and 10 mg/mL, respectively (Fig. 3).
2.3. Antimicrobial susceptibility of clinical isolates To evaluate in vitro antimicrobial activity, five common antimicrobial agents were tested against confirmed 54 clinical Mmc isolates by randomly choosing eighteen isolates from each region. The standard disc diffusion and the minimum inhibition concentrations (MICs), by micro broth dilution, methods were adopted for determination of antibiotic susceptibility following guide lines of the Clinical and Laboratory Standard Institute and Hannan (Hannan, 2000; CLSI, 2002). The antimicrobial discs (Becton Dickinson, Sparks, MD, USA) including ceftiofur sodium (10 μg), enrofloxacin (10 μg), gentamycin (10 μg), oxytetracycline (10 μg) and tylosin (10 μg), which are commonly used antibiotics for pulmonary infection in ruminants. A 10 μL of Mycoplasma culture containing approximately 1 × 107–1 × 108 CFU/mL was streaked onto PPLO agar plates. Antibiotic disc was placed gently on agar plate at recommended distance and plates were incubated in 5% CO2 at 37 °C for 24–48 h. The results were interpreted in mm according to CLSI (CLSI, 2002). For comparison, negative control disc containing DMSO (Dimethyl sulfoxide; 10%) without any antimicrobial agent was included in the study. The MICs of above mentioned antimicrobials were also determined in 96 well microtitration plates (Corning, Corning, NY, USA) as described previously (Hannan, 2000). Antimicrobials were added to the microtitration plate in triplicate to make a final concentration of 10 mg/mL in the first wells and then serially diluted by 10 folds to make further concentrations of 1, 0.1, 0.01, 0.001, 0.0001 mg/mL of each tested drug. Ten μL of the PBS washed bacterial cell (OD600 = 0.3) was pipetted to each well of the microtitration plate except the negative control which contained PPLO broth. Mycoplasma putrefaciens, obtained from Veterinary Research Institute, Lahore, was used as a positive control strain.
4. Discussion In Pakistan, caprine mycoplasmosis, which is mainly caused by important pathogenic species of Mycoplasma mycoides subsp. Capri (Mmc), is a serious threat to small ruminant production (Banaras et al., 2016; Waseem et al., 2013). The present study reported a significantly higher occurrence of Mmc in the northern zone of KPK. The possible reason of higher isolation rate from this zone might be the intense cold climatic conditions that cause stress and predispose the animals to immunosuppression and consequently make it susceptible to respiratory complications. The Mmc isolates were successfully isolated and confirmed from different clinical samples like nasal discharge, tracheal swab and lung tissues. Isolation of Mmc has also been
2.4. Statistical analysis Chi- Square test (χ2) was used for testing the association among the selected zones and sources of samples using SPSS v.19.0. The student t120
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Fig. 3. Comparative antimicrobial susceptibility of clinical Mycoplasma mycoides subsp. capri isolates against five common antibiotics using micro broth dilution method.
aminoglycosides, fluoroquinolones, tetracyclines and marcolides are common agents used in animals against mycoplasmosis (Antunes et al., 2007). Nevertheless, the microbial resistance against the common antibiotics is an emergent issue, which poses a serious health concern throughout the world (Ali et al., 2016). At the dawn of discovery of these antimicrobial agents most pathogenic organisms were highly susceptible; however, the efficacy of these antibiotics has been decreased due to development of resistance (Gautier et al., 2002). The finding of the study revealed that enrofloxacin was the most potent therapeutic agent. These results were in accordance with the findings
previously reported from tracheal and nasal discharges (Banaras et al., 2016). The highest numbers of clinical isolates were recovered from goats as compared to sheep in the current study. This may be due to the reason that goats are more sensitive to different bacterial infections including mycoplasmosis. These findings are justified by a study in which 35% and 65% of Mycoplasma spp. were isolated from sheep and goats, respectively (Al-Momani et al., 2006). Mycoplasmosis is mainly treated with different antimicrobials with various degree of success, as vaccination has not been an effective tool against this disease (Citti and Blanchard, 2013). Antimicrobials like 121
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ESBL-producing Escherichia coli from cows suffering mastitis in china contain clinical class 1 integrons with CTX-M linked to ISCR1. Front. Microbiol. http://dx.doi.org/10. 3389/fmicb.2016.01931. Antunes, N.T., Tavío, M.M., Assunção, P., Rosales, R.S., Aquili, V., De la Fé, C., Poveda, J.B., 2007. In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials. Vet. Microb. 119, 72–75. Ayling, R.D., Bisgaard-Frantzen, S., March, J.B., Godinho, K., Nicholas, R.A.J., 2005. Assessing the in vitro effectiveness of antimicrobials against Mycoplasma mycoides subsp. mycoides small-colony type to reduce contagious bovine pleuropneumonia infection. Antimicrob. Agents Chemother. 49, 5162–5165. Banaras, F., Abbas, F., Awan, M.A., Riaz, A., Khan, I.A., Mengal, M.A., Banaras, S., Murtaz-ul-Hassan Zafar, M.A., Baig, R.M., 2016. Molecular survey on the prevalence of Caprine mycoplasma in the goats of Quetta City of Pakistan. Inter. J. Vet. Sci. 5, 158–163. CLSI, 2002. Clinical and Laboratory Standard Institute, development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. CLSI document, approved guideline, 22 Wayne, Pennsylvania, USA. Citti, C., Blanchard, A., 2013. Mycoplasmas and their host: emerging and re-emerging minimal pathogens. Trends Microbiol. 21, 196–203. Economic Survey, 2013. Govt. of Pakistan an, Finance Division, Economic Advisors Wing, Ministry of Finance Islamabad, Pakistan. Gautier, A.K., Kobisch, Reinhardt, Kempf, M., 2002. In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae. Vet. Microbiol. 88, 47–58. Gelagay, A., Teshale, S., Amsaluc, W., Esayas, G., 2007. Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia. Small Rumin. Res. 70, 131–135. Hannan, P.C., 2000. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary Mycoplasma species. Vet. Res. 31, 373–395. Hotzel, H., Sachse, K., Pfützner, H., 1996. A PCR scheme for differentiation of organisms belonging to the M. mycoides cluster. Vet. Microbiol. 49, 31–43. Loria, G.R., Sammartino, R.A., Nicholas, R.D., 2003. In vitro susceptibilities of field isolates of Mycoplasma agalactiae to oxytetracycline, tylosin, enrofloxacin, spiramycin and lincomycin-spectinomycin. Res. Vet. Sci. 75, 3–7. Manso-Silvan, L., Perrier, X., Thiaucourt, F., 2007. Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group. Int. J. Sys. Evo. Microbiol. 57, 2247–2258. Mondal, D., Pramanik, A.K., Basak, D.K., 2004. Clinico-Haematology and pathology of Caprine Mycoplasma pneumonia in rain fed tropics of West Bengal. Small Rumin. Res. 51, 285–295. Nicholas, R., Ayling, R., McAuliffe, L., 2008. Respiratory diseases of small ruminants. In: Nicholas, R., Ayling, R., McAuliffe, L. (Eds.), Mycoplasma Diseases of Ruminants. CABI, Wallingford, UK, pp. 171–179. OIE, 2017. OIE-listed Diseases, Infections and Infestations in Forcein 2017. Office International DesEpizootics, World Organization for Animal Health, Paris, France (Accessed 20 May 2017). http://www.oie.int. Sadique, U., Chaudhry, Z.I., Younas, M.R., Anjum, A.A., Hassan, Z., Sajid, A., Mushtaq, M., 2012. Pathogenesis and immunohistochemical studies of caprine pleuropneumonia in experimentally infected goats. Pak Vet. J. 32, 427–731. Scott, L.C., Menzies, P.I., 2011. Antimicrobial resistance and small ruminants veterinary practice. Vet. Clin. North Am. Food Anim. Pract. 1, 23–32. Shahzad, A., Rashid, M., Muhammad, S.K., Mansur, D.A., Mohammad, I., Rashid, A., 2012. Characterization, molecular diagnosis and prevalence of caprine mycoplasmosis in different areas of Pakistan. Pak. J. Zool. 44 (2), 559–568. Waseem, S., Rashid, M., Mohammad, R., Rashid, A., Mohammad, S.K., Ghulam, A., Mohammad, I., Fayyaz, M., 2013. Prevalence, molecular diagnosis and treatment of Mycoplasma conjunctivae isolated from infectious keratoconjunctivitis affected Lohi sheep maintained at Livestock Experiment StationBahadur Nagar,Okara, Pakistan. Trop. Anim. Health Prod. 45, 737–742.
that enrofloxacin was considered as the most efficacious among the antimicrobial agents against mycoplasma infection (Hannan, 2000). Similar findings are also reported by Loria et al. (2003), they screened different antibiotics by MIC method against 24 isolates of M. agalactiae and found that enrofloxacin was the most effective agent with lowest MIC of 0.25 μg/mL. The study of Antunes et al. (2007 Antunes) also revealed that enrofloxacin and its metabolites were effective against some species of mycoides cluster like M. mycoides subspp. large colony and M. capricolum subspp. capricolum. Generally, marcolides such as tylosin is considered as the drug of choice and is extensively used in practice for the treatment of caprine, bovine and avian mycoplasmosis. However, Mycoplasma spp. has great potential to develop resistance against antimicrobial agents due to rapid mutational changes. The striking findings of this study revealed that all the local isolates were found resistant against tylosin. This could reflect indiscriminate use of tylosin. Oxytetracycline and ceftiofur sodium produced higher MIC values and lower zones of inhibition indicating intermediate resistance. In several countries of Europe, antimicrobial resistance of Mycoplasma spp. has been reported against tylosin, oxytetracycline and spectinomycin (Ayling et al., 2005). To the best of our knowledge, this is the first time comprehensive study the occurrence of clinical isolates of Mmc from diverse localities of Khyber Pakhtunkhwa. 5. Conclusions The present study concluded high occurrence of Mmc associated with cases of CCPP in small ruminants of Khyber Pakhtunkhwa province. In addition, we observed an increased resistance against commonly used antibiotics such as tylosin. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to the Pakistan Science Foundation (PSF) for providing financial assistance to conduct this study. We are also thankful to Dr. Waseem Shahzad, Veterinary Research Institute, Lahore for his technical assistance in the isolation and confirmation of local isolates. References Al-Momani, W., Halablab, M.A., Abo-Shehada, M., Katie, M., McAuliffe, L., Nicholas, R.A.J., 2006. Isolation and molecular identification of small ruminant mycoplasmas in Jordan. Small Rumin. Res. 65, 106–112. Ali, T., ur Rahman, S., Zhang, L., Shahid, M., Zhang, S., Liu, G., Gao, J., Han, B., 2016.
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