Isolation and Identification of M y c opl a sma m y coi d es Subspecies myc o ides from the Ear Canal of Cattle in Plateau State, North-Central Nigeria

Paul I. Ankeli1,4*, Mashood A. Raji4, Haruna M. Kazeem4, Moses O. Odugbo1, Nendir J. Umaru1, Idowu O. Fagbamila2, Livinus T. Ikpa2, Obinna O. Nwankiti1, Issa A. Muraina3, Pam D. Luka3 and Nicholas D. Nwankpa2,5

1Bacterial Vaccine Production Division; 2Bacterial Research Division; 3 Applied Biotechnology Division, National Veterinary Research Institute, Vom, Plateau State, Nigeria; 4 D e p ar t m ent of V e t erina r y Microbiology, Faculty of Ve t er i na r y M e di c ine, Ahmadu Bello U n i v er s i t y, Zaria, N i geri a; 5Pan African Veterinary Vaccine Centre, Addis Ababa, Ethiopia.

Abstract | This study was undertaken to isolate and identify M y c opl a sma m y coi d es su b s p e c i e s myc o ides (Mmm) from the ear canal of apparently healthy cattle in Plateau S t at e, Nigeria. One hundred and sixty six ear swab samples (n=166) were cultured from which eight (8) Mycoplasma species were isolated and characterized using conventional biochemical tests and polymerase chain reaction (PCR), respectively. Six (6) were observed to ferment glucose, reduce tetrazolium chloride and hydrolysed casein. They had no phosphatase activity, did not produce ‘film and spots’ and neither hydrolysed arginine nor urea and were preliminarily identified as members of the Mycoplasma mycoides sub-cluster which comprises Mmm and M y c opl a sma m y coi d es su b s p e c i e s capri (Mmc). Three (3) of the 8 Mycoplasma isolates were identified as Mmm using the IS1296F/R(all) and MM450/MM451 primer sets. The 574 bp product obtained by MM450/MM451 primer set gave two fragments (379, 178 bp) on digestion with Asn I restriction enzyme further confirming the isolates. This is the first report of the isolation and molecular identification of Mmm from the ear canal of cattle in Nigeria. It is recommended that samples from the ear canal be considered when Mmm isolation is intended so as to improve the chance of recovery.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | June 08, 2016; Accepted | July 22, 2016; Published | August 29, 2016

*Correspondence | Paul I. Ankeli, Bacterial Vaccine Production Division, National Veterinary Research Institute, Vom, Plateau State, Nigeria; Email: drpaulidokoankeli@yahoo.com

Citation | Ankeli, P.I., O. Fagbamila, L.T. Ikpa, I.A. Muraina, P.D. Luka, M.A. Raji, H.M. Kazeem, M.O. Odugbo, N.J. Umaru, I.O.O. Nwankiti and N.D. Nwankpa. 2016. Isolation and identification of M y c opl a sma m y coi d es subspecies myc o ides from the ear canal of cattle in Plateau State, North-Central Nigeria. Veterinary Sciences: Research and Reviews, 2(2): 52-59.

DOI | http://dx.doi.org/10.17582/journal.vsrr/2016.2.2.52.59

Introduction

M ycoplasma mycoides subspecies mycoides (Mmm), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, belongs to the genus Mycoplasma in the class Mollicutes (Westberg et al., 2004; Pilo et al., 2007; Li et al., 2009). It was the first mycoplasma to be described (Nocard and Roux, 1898) and has been studied extensively ever since (Ta r d y et al., 2011). Phylogenetically, Mmm is a member of the Mycoplasma mycoides cluster which are pathogens of ruminants that include M. mycoides subsp. capri (Mmc), M. capricolum subsp. capripneumoniae (Mccp), M. capricolum subsp. capricolum (Mcc) and Mycoplasma leachii (Manso-Silvan et al., 2009). The se m y copl as m as a re dif f i cult to dif f e r enti ate b ecause of th eir genotypic and phenotypic relatedness (Tardy et al., 2009). However, the precise identification of Mmm is critical as it causes CBPP which is currently regarded as the most important cattle

disease in sub-Saharan Africa since the eradication of rinderpest (Thiaucour t et al., 2000; Gull et al., 2013).

Several genomic techniques have been developed which enable the identiﬁcation of Mycoplasma species (McAuliffe et al., 2005; Stakenborg et al., 2005) and robust diagnostic systems based on po l y m er a se c h ain re a c t ion (PC R) are available for the rapid detection, identification and differentiation of members of the Mycoplasma mycoides cluster and the specific identification of Mmm (Nicholas et al., 2008). Polymerase chain reaction (PCR) has enhanced a better resolution in the diagnosis and differentiation of mycoplasmas compared to conventional methodologies of culture and serology (Woubit, 2008) and thereby made detection and identification of Mmm more sensitive and reliable (Bashiruddin et al., 1994; Miles et al., 2006).

Isolation, though labour intensive and limited in sensitivity, remains the gold standard for the diagnosis of mycoplasma infections (Egwu et al., 2012; OIE, 2014). Several Mycoplasma species have been isolated from the ear canal of cattle some of which include M. b o vis (Maeda et al., 2003; Francoz et al., 2004; Foster et al., 2008; Santos et al., 2012), M. alkalescens (Lamm et al., 2004; Nwankpa, 2008; Santos et al., 2012), M. arginini, M. bovirhinis, M. capricolum, M. conjunctivae, M. mycoides subsp. capri (Santos et al., 2012) and M. leachii (Nwankpa, 2008) but from available literature, Mmm isolation from the ear canal of cattle has not been documented. The ear canal is an unusual anatomical site for Mmm recovery, thus the aim of our study was to isolate and identify Mmm from the ear canal of apparently healthy cattle in Plateau State, North Central Nigeria (Figure 1).

Materials and Methods

Study Area

Plateau State is located in the Middle Belt zone of Nigeria and lies between Latitudes 8º and 10º North and Longitudes 7º and 11º east. It shares common boundaries with four of the thirty six states of the Federation. Though situated in the tropical zone, the climate of the State is the nearest equivalent of temperate climate with mean temperatures of 18ºC and maximum of 30ºC, while mean annual rainfall varies between 1317.5 mm in the Southern part and 1460.0 mm on the high Plateau. Cattle are the major source of meat supply in Plateau State.

Sample Collection and Processing

A total of 166 swab samples of the ear canal were collected from live cattle in 6 markets (n = 106) and 12 farms (n = 60) respectively, from May 2013 – March 2016. The swabs were cut directly into Bijou bottles containing 3 ml mycoplasma broth culture medium (used as transport medium) and screwed tight. The mycoplasma broth medium contained 21g/l PPLO broth powder (Difco®), 20% de-complemented horse serum (Quad Five®), 10% fresh yeast extract, 0.2% glucose (Fluka®), 0.4% sodium pyruvate (MRS Scientific®) and 0.04% ampicillin (Quadrant®), was prepared according to the methods described by Freundt (1983) and Provost et al. (1987). Samples were kept on ice and transported to the Mycoplasma Laboratory, National Veterinary Research Institute, Vom, Plateau State, Nigeria for processing.

Cultivation and Preliminary Identification of Mycoplasma Species

Ear swab samples in transport medium (mycoplasma broth) were processed for mycoplasma isolation as described by the OIE manual (2014). Briefly, a four ten-fold serial dilution (10-1 to 10-4) in mycoplasma broth medium was carried out. A 100 µl of the last dilution (10-4) was inoculated into Bijou bottle containing 1.9 ml of mycoplasma broth medium and incubated at 37°C in aerobic conditions for 3 days later and sub-cultured onto mycoplasma agar plates. Agar plates were incubated at 37°C in 5% carbon dioxide under maximum humidity and monitored for evidence of mycoplasma-like (“fried egg”) colonies every 3 days for a period of 2 weeks using a stereomicroscope at x35 magnification. Isolated colonies having classical ‘fried egg’ appearance with dense centers were triple-cloned, mixed with equal volume of glycerol (used as a cryo-preservative) and stored in duplicates at -20ºC and -80ºC, respectively.

For the preliminary identification of the isolates; the growth on mycoplasma media without serum as well as without antibiotic, and the classical biochemical tests which include glucose fermentation, arginine hydrolysis, tetrazolium reduction, phosphatase production, and urea and casein hydrolysis as well as production of ‘film and spots’ were carried out on each of the isolates as described by Poveda (1998).

Genome Amplification of Mmm isolates

DNA was extracted from isolates using ZR F u n g a l/B a c t e r i al D N A M i n i P r e p™ kit (Zymo Research Corp., Irvine, CA, USA) according to manufacturer’s instructions. Lyophilized T1/44 vaccine (NVRI, Vom Nigeria) was used as control for this study. The presence of Mmm DNA was first diagnostically confirmed by the amplification of 1.1 kbp and 574 bp fragments using the protocol described by Miles et al. (2006) and Bashiruddin et al. (1994), respectively. The 574 bp PCR products were further digested by Asn I as described by Bashiruddin et al. (1994) to obtain two fragments (379, 178 bp) for Mmm and three fragments (220, 178, 153 bp) for Mmc, respectively. PCR products obtained were resolved on 1.0% agarose gel impregnated with ethidium bromide (0.5ug/ml) at 130 volts for 50 minutes and visualized on Bio Rad Gel Doc™ XR+ (BioRad, CA, USA) system.

Results

Mycoplasma Isolation

A total of 166 ear swab samples collected for Mmm

Table 1: Biochemical characteristics of mycoplasmas isolated from the ear canal of cattle in Plateau State

| Biochemical tests |  |  |
Number of isolates recovered (%) | GF | AH | TR | PP | UH | CH | FS | Remarks
6 (75.0%) | + | - | + | - | - | + | - | Mmm/Mmc
2 (25.0%) | - | + | - | + | - | - | - | M. alkalescens

GF: glucose fermentation; AH: arginine hydrolysis; TR: tetrazolium reduction; PP: phosphatase production; UH: urea hydrolysis; CH: casein hydrolysis; FS: film and spot production

isolation, 8 yielded mycoplasma-like isolates when cultured on mycoplasma media. Mycoplasma-like growth observed in broth varied from a homogeneous cloudiness to slight turbidity, produced w ithin 3–4 days, occasionally with a silk y, fragile filament when broth media bottles were swirled. The growth observed on mycoplasma agar were characteristically small, circular, and convex with raised dark centres and lighter peripheries giving a ‘fried-egg’ appearance (Figure 2) as described by the OIE Manual (2014).

Preliminary Identification of the M yc o plas m a Isolates

Gro wth of Mycoplasma isolates in serum-free and in antibiotic-free mycoplasma media. None of the isolates grew in serum-free mycoplasma medium (sterol dependency test) and all maintained the ‘fried egg’ appearance of colonies on mycoplasma agar medium without antibiotic, after 3 passages. They were thus designated mycoplasmas belonging to the family Mycoplasmataceae (Mycoplasma or Ureaplasma species), ruling out Acholeplasma species and L-form bacteria respectively, and giving an overall Mycoplasma species isolation rate of 4.82% from the ear canal.

Biochemical Characterization of Mycoplasma Species

Biochemical characterization of the 8 mycoplasma isolates indicated that 6 fermented glucose, reduced tetrazolium chloride and hydrolysed casein. They did not hydrolyze arginine nor urea and neither produced phosphatase and ‘film and spots’, and were presumptively identified as members of the Mycoplasma mycoides sub-cluster (either Mmm or Mmc). Both Mycoplasma subspecies share these biochemical properties even though Mmc is a pathogen mostly of small ruminants. The other 2 mycoplasma isolates only hydrolysed arginine and produced phosphatase and were presumptively identified as M. alkalescens. Ureaplasma and other urea-utilizing Mycoplasma species were ruled out as none of the mycoplasma isolates hydrolysed urea (Table 1).

Molecular Identification of Mmm using PCR

A total of 8 mycoplasma isolates were subjected to PCR for identification. Out of them, 6 isolates presumptively identified by biochemical characterization as members of the Mycoplasma mycoides sub-cluster (Mmm and Mmc), 3 were positively identified as Mmm by PCR specific primers as described by Miles et al. (2006). The 6 presumptive Mycoplasma mycoides sub-cluster members gave 574 bp (Figure 3) following amplification by M450/M451 (Bashiruddin et al., 1994). Out of 6, three each were identified as Mmm and Mmc by producing two and three fragments each with the AsnI restriction enzyme (Bashiruddin et al., 1994). The remaining 2 mycoplasma isolates that hydrolysed arginine and produced phosphatase were negative for both Mmm and Mmc specific PCR and were therefore excluded from the study (Figure 4; Table 2).

Table 2: Isolation rate of Mmm from ear canal of cattle in Plateau State

Sample type | Number examined | Number positive | Isolation rate (%)
Cattle markets | 106 | 2 | 1.89
Cattle farms | 60 | 1 | 1.67
Total | 166 | 3 | 1.81

Discussion

The ear canal has now been included as one of the recommended sampling sites for pathogenic mycoplasmas in small ruminants (DaMassa and Brooks, 1991; Nicholas and Baker, 1998) and cattle (Santos et al., 2012). In view of the well-documented recoveries of mycoplasmas other than Mmm from the ear canal of cattle, its presence in the ear canal is therefore not unlikely. However, information on the isolation of Mmm from the ear canal of cattle is not available in literature. This might be attributed to the fact that previous studies were carried out in CBPP-free regions of the world (Chazel et al., 2010; Tardy et al., 2011; Santos et al., 2012), or due to the non-inclusion of culture for Mycoplasma species in the investigation of most auricular diseases (Vestweber, 1999; Francoz, 2004). Alternatively, the dearth of information on the isolation of Mmm from the ear canal of cattle can also be as a result of the collection of too few ear canal samples for Mycoplasma species isolation considering that the predilection site for most mycoplasmas is the respiratory system (Thiaucourt et al., 2004); other than Mmm from the ear canal.

Considering the ability of other ruminant mycoplasmas to colonise the ear canal, ear involvement by Mmm is not unlikely. Several hypotheses can be suggested to explain the occurrence of Mmm in the ear canal. Firstly, its extension from the pharynx via the Eustachian tube to the middle ear as described by W alz et al. (1997), Maeda et al. (2003) and Lamm et al. (2004) for the pathogenesis of otitis due to Mycoplasma bovis infection in cattle. Secondly, the my c opl asma e m ia d e v e l oped during acute infection which may involve other organ systems (Gull et al., 2013) including the auditory organs of the sensory system. The fact that viable Mmm have been recovered from organs other than those of the pulmonary system provides considerable evidence that this mycoplasma can indeed disseminate from the respiratory tract occasionally (Thiaucourt et al., 2004). Additionally, ear mites have also been associated with potentially pathogenic Mycoplasma species of cattle in a CBPP-free country (Santos et al., 2012) as well as with pathogenic Mycoplasma species of small ruminants (Francoz et al., 2004; Mercier et al., 2007). The association of ear mites with Mmm in the ear canal of cattle in enzootic areas is likely, although in our study ear mite infestation was not investigated. Alternatively, the contamination of the ear canal by Mmm in CBPP-infected herds is also possible as previously reported by Corrales et al. (2007) and Arcangiolo et al. (2012) for Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in goats and Mycoplasma bovis infection in calves, respectively.

The Mmm isolation rate in this study was low (1.81%). This may be attributed to collection of samples from apparently healthy cattle which is in agreement with Duarte et al. (2003) who reported a significantly lower isolation rate of Malassezia species from the external ear canal of apparently healthy cattle compared to those with otitis. It is possible that if apparently sick animals presenting classical signs of CBPP had been sampled, the isolation rate might have been higher. The low isolation rate may also be due to the inferiority of ear canal swabbing compared to ear flushing technique in goats as reported by Ribeiro et al. (1997). On the other hand, the low isolation rate may also be due to the ear canal being an unusual anatomical site for mycoplasma isolation as reported in goats by Cottew and Yeats (1981) and (1982). Perhaps the organism was present in little amounts in ear samples as it is a causative pathogen of a respiratory disease. Also, the low positivity may be due to the study design, unwillingness of herdsmen to allow for sampling and insecurity during the study period. Additionally, constraints such as the heavy presence of antibiotics in the clinical samples, contamination of samples by bacteria and overgrowth by less important but more exuberant mycoplasmas, and the complex requirements for growth may have affected Mmm isolation rate (Nicholas et al., 2008; Hassan et al., 2011) in this study because the organism is labile and often present in little quantities (Gedlu, 2004).

In this present study, Mmm from ear canal swab samples of apparently healthy cattle in 2013–2016 were detected by PCR in this study. Out of 6 isolates identified as members of the Mycoplasma mycoides sub-cluster, 50% (3/6) were confirmed as Mmm following the OIE (2014) guideline. These findings are similar to that of Bashiruddin et al. (1994) and Miles et al. (2006).

Conclusion

In conclusion, the present study is the first in assessing the presence of Mmm from the ear canal of cattle in Plateau State, Nigeria. This is of major significance as it implies that the ear canal can be included as a sample collection site for Mmm recovery compared to the nasal swab samples which are more prone to contamination by commensal and opportunistic bacteria. The occurrence of Mmm in apparently healthy cattle in this study also indicates that it is part of the normal flora of the ear canal.

Acknowledgement

The authors thank the NVRI, Vom for funding this project. The authors also appreciate members of the Mycoplasma Research Group (Sunday Kigbu, Mary Peter Gyang, Emmanuel Damter, Salma Maichibi and Rosemary Goji) and staff of the Applied Biotechnology Division (Olajide Owolodun, Nneka Chima and Anvou Jambo), all of the NVRI for their technical support.

Conflict of Interest

The authors declare that there is no conflict of interest.

Authors’ Contribution

MAR, HMK and PIA conceived and designed the study. PIA, NJU, IOF, LTI, IAM, PDL and OON collected and analysed samples. MAR, HMK, PIA, PDL, MOO and NDN drafted the manuscript. All authors read and approved the final manuscript for submission.

References

- • Arcangiolo, M.A., Froux, W., Poumarat, F., Le Grand, D. Case reports: Mycoplasma bovis otitis in French dairy calves. Revue de Médecine Vétérinaire, 2012; 163 (6): 184-286.
- • Bashiruddin, J.B., Taylor, T.K., Gould, A.R. A PCR-based test for the specific identification of Mycoplasma mycoides subspecies mycoides SC. Journal of Veterinary Diagnostic Investigation, 1994; 6: 428-434. http://dx.doi.org/10.1177/104063879400600405
- • Chazel, M., Tardy, F., Le Grand, D., Calavas, D., Poumarat, F. Mycoplasmoses of ruminants in France: recent data from the national surveillance network. BMC Veterinary Research, 2010; 6: 32. http://dx.doi.org/10.1186/1746-6148-6-32
- • Corrales, J., Esnal A., De la Fe, C., Sánchez, A., Assunçao, P., Poveda, J., Contreras, A.. Contagious agalactia in small ruminants. Small Ruminant Research, 2007; 68: 154-166. http://dx.doi.org/10.1016/j.smallrumres.2006.09.010
- • Cottew, G.S., Yeats, F.R. Occurrence of mycoplasmas in clinically normal goats. Australian Veterinary Journal, 1981; 57: 52–53.223
- • Cottew, G.S., Yeats, F.R. Mycoplasmas and mites in the ears of clinically normal goats. Australian Veterinary Journal, 1982; 59: 77–81. http://dx.doi.org/10.1111/j.1751-0813.1982.tb02731.x
- • DaMassa, A.J., Brooks, D.L. The external ear canal of goats and other animals as a mycoplasma habitat. Small Ruminant Research, 1991; 4: 85–93. http://dx.doi.org/10.1016/0921-4488(91)90055-U
- • Dedieu, L., Mady, V., Lefèvre, P.C. Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. mycoides S.C. (contagious bovine pleuropneumonia agent). Veterinary Microbiology, 1994; 42(4): 327-39. http://dx.doi.org/10.1016/0378-1135(94)90064-7
- • Duarte, E. R., Batista, R. D., Hahn, R. C., Hamdan, J. S. Factors associated with the prevalence of Malassezia species in the external ears of cattle from the state of Minas Gerais, Brazil. Medical Mycology, 2003; 41: 137–142. http://dx.doi.org/10.1080/mmy.41.2.137.142
- • Eg wu, G.O., A damu, M., M s h elia, G.D., B ukar-Kolo, Y.M. A s us t a i n a b le l a b o r atory a p p r o a ch for c o n t a g i ou s bo v i ne ple u r o pneumo nia (CB P P) mo n itoring in N i g e ria: c om p arison be t w e en t wo s e r o l og i c al te s ts in an en d e mic area c o m pli m e nted with p ost m ortem le sio n s. A fr i c an Jo u r n al of M i c r obio l o gy R esea rch, 2012; 6 (3 0): 5 89 0-589 5.
- • F oste r, A.P., N a ylo r, R.D., H o wi e, N.M., Nicholas, R.A.J., A ylin g, R.D. Mycoplasma b o vis and otitis in dairy calves in the UK. The Veterinary Journal, 2009; 179 (3): 455-457.
- • Francoz, D., Fecteau, G., Desrochers, A. Fortin, M. Otitis media in dairy calves: A retrospective study of 15 cases (1987 to 2002). Canadian Veterinary Journal, 2004; 45: 661-666.
- • Freundt, E.A. 1983. The culture media for classic m ycoplasmas. In: Raz in, S.G. and Tully, J. (eds). Methods in Mycoplasmology, Academic Press New York, USA. pp. 127-135. http://dx.doi.org/10.1016/B978-0-12-583801-6.50029-9
- • Gedlu, M.G. 2004. Serological, clinical and participatory epidemiological survey of contagious bovine pleuropneumonia in Somali Region, Ethiopia. An MSc thesis submitted to the Faculty of Veterinary Medicine, Addis Ababa, Debre Zeit, Ethiopia.
- • Gevers, D., C o h an, F.M., La wrence, J.G., Spratt, B.G., Co eny e, T., Feil, E.J., Stackebrandt, E., de Peer, Y.V., Vandamme, P., Thompson, F.L., Swings. J. O pin ion: Re-eval uating prokaryotic species. National Reviews in Microbiology, 2005; 3: 733-739. http://dx.doi.org/10.1038/nrmicro1236
- • Gull, T.B., F rench, R.A., Gor ton, T.S., Bur rage, T.G., Prozesky, L., Gear y, S.J. and Adams, G.L. Models of contagious bovine pleu ropneumonia: Eva luation of two n ov e l s t r a i n s. T he O pen V e t e r i n a ry S c i e n ce Jo u r n a l, 2013; 7: 23-33. http://dx.doi.org/10.2174/1874318820130731002
- • Hassan, W.H., El-S hab rawy, M.A., Haki m, A.S., Abueln aga, A.S.M., Samy A.A. and Sadek E.G. Co m paris o n between m olecular and classical t e c h n iq u es f o r i d enti f icati o n of M y cop l a s ma species i s o lat e d from mastitic r u m inant s. Journal of Ame rican Scie n ce, 2011; 7(1): 506-613.
- • Lamm, C.G., Munson, I., Thu r mond, M.C., Bar r, B.C. and George, L.W. M y coplasma otitis in California calves. Journal of Veterinary Diagnostic Investigation, 2004; 16: 397–402. http://dx.doi.org/10.1177/104063870401600505
- • Li, Y., Xin, J., Gao, Y., Zhang, J., Nicholas, R.A.J., Lin, Y. Strains of Mycoplasma mycoides subsp. mycoides Small Colony type isolated in China between 1953 and 1960 show close similarity to strains of the Africa/Australia cluster. The Veterinary Journal, 2009; 179: 137–141. http://dx.doi.org/10.1016/j.tvjl.2007.08.021
- • Maeda, T., Shibaha r a, T., Kimura, K., Wada, Y., Sato, K., Imada, Y., Ishikawa, Y. K adota, K. Mycoplasma b o vis-associated suppu ra t i v e otitis media and pneumonia in bull calves. Journal of Comparative Pathology, 2003; 129: 100–110. http://dx.doi.org/10.1016/S0021-9975(03)00009-4
- • M a n s o-S ilv a n, L., V il e i, E. M., S a c h se, K., D jo r d j e vi c, S. P., T h i a u c o u r t, F., F r ey, J. M y c o p l a s m a l e a c h i i s p. no v. a s a n e w s p ec i e s d e s ig n a t i o n f o r M y c o pla s m a s p. b o vi n e g r o u p 7 of L e a c h, a nd r ec l ass ifi c a t i on of M yc o p l a s m a m y c o i d e s s u b s p. m y c o id e s LC as a s e ro v ar of M yc o p l a s m a m y c o id e s s u b s p. c ap r i. In te rna t i o na l J o u r na l o f S y s t e ma t i c an d E v o l u t i o nar y Mi c r o bi o l o g y, 2009; 59: 13 5 3-1 3 58. http://dx.doi.org/10.1099/ijs.0.005546-0
- • McAuliffe, L., Ellis, R., La wes, J., Ayling, R.D., Nicholas, R.A.J. 16S rDNA and DGGE: a sin g le g eneric test for detecting and differentiating Mycoplasma species. Journal of Medical Microbiology, 2005; 54: 1–9. http://dx.doi.org/10.1099/jmm.0.46058-0
- • Mercier, P., Pellet, M.P., Morignat, E., Calavas, D., Poumarat F. Prevalence of mycoplasmas in external ear canal of goats: influence of the sanitary status of the herd. Small Ruminants Research, 2007; 73, 296-299. http://dx.doi.org/10.1016/j.smallrumres.2007.01.018
- • Miles, K., Chu r ch w ard C.P., Mcauliffe L., Ayli ng R.D., Nicholas R.A. Ide ntification and differentiation of Europ e an and African/Australian strains of Mycop l a s ma m y coi d es subspecies my c o i de s small-colony type usi ng polymerase chain reaction analysis. Journal of Veterinary Diagnostic Investigation, 2006; 18: 168–171. http://dx.doi.org/10.1177/104063870601800205
- • Nicholas, R.A.J. and Baker, S.E. 1998. Recovery of mycoplasmas from ani mals. In: Miles, R.J. and R.A.J. Nicholas (eds.), Mycoplas m a Protocol s. Hum ana Press, Totowa, USA. pp. 37-44. http://dx.doi.org/10.1385/0-89603-525-5:37
- • Nicholas, R.A.J., A yling R., M c A uli ffe L. 2 0 08. Contagious bo vine pleuro pn e u m onia. In: Myc op l a s m a Diseases of R u mi n an ts. R. Nic h olas, R. Ayling a nd L. M c A uliffe (eds) C ABI P u blishing, W allin g f ord, UK. p p. 69-97. http://dx.doi.org/10.1079/9780851990125.0000
- • No c a r d, E., R o u x, E. L e m i c r ob e d e l a p é r i pn e um on i e. A nn al s d e l’in st i t ut e P a s te u r, 1898; 1 2: 240-2 62.
- • Nwankpa, N.D. 2008. Serological and molecular studies of Mycoplasma mycoides mycoides Small Colony in Northern Nigeria. Ph. D thesis submitted to the Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine University of Nigeria, Nsukka.
- • Office International des Epizooties (OIE). 2014. Contagious bovine pleuropneumonia. OIE Terrestrial Manual. pp. 1-16.
- • Pilo, P., Frey, F., Vilei, E.M. Molecular mechani s ms of pathogenicity of M y co p lasma my c oides su bsp. my c oides SC. Veterinary Journal, 2007; 174 (3): 513–521. http://dx.doi.org/10.1016/j.tvjl.2006.10.016
- • Poveda, J.B. 1998. Biochemical characteristics in Myc o plasma identification. In: Miles, R. J., R. A. J. Nicholas (eds.), Myco p las m a Protocol s. Hum ana Press. Totowa, USA, pp: 69-78. http://dx.doi.org/10.1385/0-89603-525-5:69
- • Provost, A., Perreau, P., Breard, A., Le Goff, C., Martel, J. L., Cottew, G. S. Péripneumonie contagieuse bovine. Revue Scientifique et Technique, Office International des Epizooties, 1987; 6(3): 565-679. http://dx.doi.org/10.20506/rst.6.3.306
- • Ribeiro, V.R., Nascimento, E.R, Faccini, J.L.H., Nascimento, M.G.F., Lignon, G. B. An improved method for the recovery of mycoplasmas from the external ear canal of goats. Journal of Veterinary Diagnostic Investigation, 1997; 9: 156-158. http://dx.doi.org/10.1177/104063879700900208
- • Santos, S.B., Nascimento, E.R., Faccini, J.L.H., Barreto M.L., Mota R.A. Associação entre Mycoplasma spp. e ácaros do conduto auditivo de bovinos [Mycoplasmas species associated with the ear mites in cattle]. Pesquisa Veterinária Brasileira, 2012; 32(4): 293-296. http://dx.doi.org/10.1590/S0100-736X2012000400004
- • Santos, S.B., Nascimento, E.R., Faccini, J.L.H., Barreto, M.L., Pereira, V.L. Potentially pathogenic mycoplasmas in the external ear canal of clinically normal cattle in Southeast Brazil: first report. Brazilian Journal of Microbiology, 2009; 40 (3): 455-457.
- • Stakenborg, T., Vicca, J., Buta y e, P., Maes, D., De Baere, T., Verhelst, R., Peeters, J., de Kruif, A., Haesebrouck, F., V aneechoutt e, M. E v alu a tion of ampliﬁed rDNA restric tion analysis (ARDRA) for the identiﬁcation of Mycoplasma species. BMC Infectious Diseases, 2005; 5: 46. http://dx.doi.org/10.1186/1471-2334-5-46
- • Tardy, F., Gaurivaud, P., Manso-Silvan, L., Thiaucourt, F., Pellet, M.P., Mercier, P., Le Grand, D., Poumarat, F. Extended surveillance for CBPP in a free country: challenges and solutions regarding the potential caprine reservoir. Preventive Veterinary Medicine, 2011; 101: 89-95. http://dx.doi.org/10.1016/j.prevetmed.2011.04.017
- • Tardy, F., Maigr e, L., C i tti C., P ou m a r a t, F. 2009. Fuzzy b oun daries of the « M y coi d es » taxa: a call f or new diagnos is s t r a t e g i es? M y c opla s m ology - a r e vi e w of d e v e lop m e n t s ov e r t he la s t d e c a d e. G r an C ana r i a, C ana r y Is land s, Spa i n. pp. 36.
- • Thiaucourt, F., van der Lugt, J.J., Provost, A. 2004. Contagious bovine pleuropneumonia, in J.A.W. Coetzer & R.C. Tustin (eds). Infectious diseases of livestock (2nd edn), Vol. 3, Oxford University Press, Cape Town. pp. 2045-2059,
- • T h i a u c o u r t, F., Y a y a, A., W e s onga, H., Hü b s c hl e, O.J., T ulas n e, J.J., Pr o v o s t, A. C o n t a gi o us o vi ne p l e u r o p n e u m o n i a. A re ass e ss m e nt of t he effi c a cy of v a cc i n es u s ed in Afr i c a. Annals of the New York A c ademy of Sciences, 2000; 9 1 6: 71-80. http://dx.doi.org/10.1111/j.1749-6632.2000.tb05276.x
- • Vestweber, J.G. Otitis media/interna in cattle. Compendium on continuing education for the practicing veterinarian, 1999; 21: S34–S38.
- • Walz, P.H., Mullane y, T.P., Render, J., Walker, R.D., Mosser, T., Baker, J.C. Otitis media in reweaned Holstein dairy calves in Michigan due to Mycoplasma b o vis. Journal of Veterinary Diagnostic Investigation, 1997; 9: 250–254. http://dx.doi.org/10.1177/104063879700900305
- • Westberg, J., Persson, A., Holmberg, A., Goesmann, A., Lundeberg, J., Johansson, K.E., Pettersson, B., Uhlen, M. The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Research, 2004; 14: 221-227. http://dx.doi.org/10.1101/gr.1673304
- • Woubit, S. 2008. Utilisation d’une séquence complète du génome d’une souche de Mycoplasma mycoides subsp. mycoides LC pour la mise au point de tests de diagnostic, application à l’expression de gènes hétérologues. Le titre de Doctoeur de L’Institut National Polytechnique de Toulouse PhD Ecole doctorale: Sevab Science Veterinaire.