Research ArticleIsolation and Characterization of Mycoplasma mycoidesSubspecies capri from Milk of Natural Goat Mastitis Cases
Vijay Kumar1 Rajneesh Rana2 Somya Mehra1 and Pramod Kumar Rout1
1 Central Institute for Research on Goats Makhdoom PO Farah Mathura 281122 Uttar Pradesh India2Division of Bacteriology and Mycology Indian Veterinary Research Institute Izatnagar Bareilly 243 122 Uttar Pradesh India
Correspondence should be addressed to Rajneesh Rana rajneeshrana01yahoocom
Received 4 April 2013 Accepted 24 April 2013
Academic Editors P Butaye J Cabaret and I Nsahlai
Copyright copy 2013 Vijay Kumar et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Association ofMycoplasmamycoides subspecies capri (Mmc) with natural goat mastitis has been studied earlier largely by detectingtheMmc DNA using molecular methods However report on detection of cultivableMmc isolates from natural goat-mastitis milkis still very rare In this study Mmc was isolated from milk samples (119899 = 171) of goats with or without clinical signs of mastitisMmc isolates were further characterized by biochemical and species-specific PCR methods Intra species strain variation was alsostudied by 16S amplified rDNA restriction analysis (16S ARDRA)The study recovered a total of 6Mmc isolates (35)Three typesof intraspecies variants among the recoveredMmc isolates were found by 16S ARDRA The study concluded that Mmc may be anetiological agent of mycoplasmal mastitis in Indian goat herds
1 Introduction
Mycoplasma mycoides subsp capri (Mmc) belongs to theldquoMycoplasma mycoidesrdquo cluster (M capricolum subsp capri-colum M capricolum subsp capripneumoniae M mycoidessubsp mycoides large colony type (LC) M mycoides subspmycoides small colony type (SC) Mycoplasma spp bovinegroup 7 and Mmc) and is reported to cause a pattern ofdisease (mastitis arthritis keratoconjunctivitis and pleurop-neumonia) in goats similar to those induced by the rest ofthe species of the mycoides cluster and other mycoplasmasnamely M agalactiae and M putrefaciens [1 2] Mastitis isone of themanifestations of contagious agalactia (CA) [3] andis characterized by clinical signs like heat pain swelling andredness in the udder besides alteration in milk (clot flakesdiscoloration and reduction or complete cessation of milkyield) CA is prevalent in several regions of the world [4] bycausing high morbidity (261ndash100) in adult goats and 365to 100 in kids [5] along with 25 and 90 mortality inadult goats and kids respectively [6] In goat-rearing units theeconomic loss may reach up to 15ndash20 [4]
AlthoughM agalactiae is known as the classical etiologi-cal agent of CA andormastitis other species of themycoides
cluster have also been found to be associated with goat mas-titis in different countries [7ndash9] Very recently Amores et al[9] have detected Mmc using polymerase chain reaction(PCR) from bulk tank milk which was collected from goatsexhibiting clinical signs of mastitis from a CA endemic areaHowever there is no report about the isolation ofMmc fromnatural goatrsquos mastitis except for the experimental study ofMisri et al [2] and DrsquoAngelo et al [10]
In view of the dearth of information on the associationofMmc with goat mastitis on culture bases the present studywas carried out to determine the involvement ofMmc as wellas intraspecific strain variation by isolation characterizationbased on species specific PCR and 16S amplified rDNArestriction analysis (16S ARDRA) pattern
2 Materials and Methods
21 Classification of Goats for Sampling Goats exhibiting theclinical signs of mastitis that is swollen udder with painsecreting altered milk fever lethargy and labored breathingand goats either living in close proximity to goats sufferingfrommastitis or in herds having a history of CAwere selected
lowastMmcM mycoides subsp capriMmm LCM mycoides susp mycoides large colony typeM agM agalactiaeMccM capricolum subsp capricolumMbg7M bovine group 7
for sampling Goats not exhibiting clinical mastitis weretaken as asymptomatic ones and suspected for carriers ofmycoplasmas
22 Goat Herd Sample Collection and Isolation of Mycoplas-mas A total of 171 goats were sampled for milk from fivedifferent goat herds of Mathura region (CIRG MakhdoomJhandipur Chattar Singh Ka Nagla Keetham and Agra)that facing the problems of CA andor mastitis Out of 171milk samples 102 were from clinical mastitis and 69 fromasymptomatic goats Milk samples were collected asepticallyin sterile vials that is before milking affected teats werecleaned with 70 ethanol and then the first 2-3 streams werediscarded
Isolation of mycoplasmas from milk samples was per-formed as described by Carmichael et al [11] with slightmodifications Briefly milk samples were inoculated intoliquid Hankrsquos balanced salt solution (HBSS-L pH 76ndash78)medium and incubated at 37∘C under 5 CO
2for up to 14
days and subsequently transferred on solid Hankrsquos balancedsalt solution (HBSS-S) medium The probability of L phasevariants was ruled out by forward and reversal passages inthe artificial medium
23 Biochemical Characterization A preliminary characteri-zation of the isolates was performed by digitonin sensitivityand growth inhibition tests as per the method describedelsewhere [12] and Giemsa method of staining This wasfollowed by biochemical tests namely glucose fermentationphosphate reduction gelatin hydrolysis and film and spotformation test as described earlier [13]
24 Confirmation of Isolates by PCR Genomic DNA ofthe isolates was extracted from late exponential growthphase using the phenol-chloroformmethod described by vanKuppeveled et al [14] and purity and concentration of DNAwas checked on 07 agarose gel and spectrophotometricanalysis according to Sambrook et al [15]
Mycoplasma isolates were confirmed by employingMmc-specific PCR and the presence of other mycoplasmas wasruled out by conducting the respective species-specific PCRsaccording to respective protocols described elsewhere asreferred in Table 1 The Qiagen PCR core kit was used toperform all the PCRs and consequent PCR products werechecked on 2 agarose gel
25 Characterization of Intraspecies Strain Variation Using 16SARDRA The 16S rDNA of all isolates was amplified by usingthe universal primer pair pA (51015840-AGAGTTTGATCCTGG-CTCAG-31015840) and pH (51015840-AAGGAGGTGATCCAGCCGCA-31015840) for 30 cycles (20 sec 94∘C 15 sec 57∘C and 30 sec72∘C) using Qiagen PCR core kit as per Edwards et al[21] The resultant amplicon (1500 bp) was purified by usingpurification kit (Bangalore Genei India) It was subsequentlydigested with restriction enzymeAlu I (Fermentas sequenceAGandCT) and the restriction fragments were separated on 3NuSieve 3 1 agar by using the method of Stakenborg et al[22]
3 Results and Discussion
Out of 171 clinical and asymptomatic samples a total of45 samples showed fine turbidity and pH shift (acidic)imparting a yellow color to the broth medium within 3 to10 days indicating the mycoplasma growth After followingthe protocol of 4-5 reversal and 3-4 forward passages thepossibility of ldquoL phase variantrdquo was ruled out Only 6 (35)samples yielded colonies of 1 to 2mm size exhibiting typicalfried egg appearance on HBSS-S medium Their growthcharacteristics were indicative of the mycoplasmas Of sixisolates 5 were recovered from clinical mastitis milk whereasone (isolate number 6) was from subclinical mastitis milkThese growth evidences were in accordance with Razin andFreundt [23] and Sori et al [24] In the study the isolationrate (35) was found to be in agreement with Ikhloea et al[25] who obtained similar results of 37 to 11 however our
ISRN Veterinary Science 3
PC 1 NC 2 3 4 5 6
M
Figure 1 M mycoides subsp Capri-specific PCR exhibiting 195 bpamplicon M 100 bp DNA ladder PC positive control (M mycoidessubsp capri PG-3) NC negative control Lanes 1ndash6 respectiveisolates
isolation rate seems to be quite low in contrast to the 25 to71 obtained by Gil et al [26]
All the isolates showed purplish-pinkish coccobacillarybodies with pleomorphic shape and size upon Giemsa stain-ing The isolates passed filtration test through 045120583m filterand found to be sensitive to digitonin Biochemical testsnamely glucose fermentation and gelatin hydrolysis testsgave positive results while film and spot formation test andphosphatase test were negative for all isolates The isolatesexhibited positive growth inhibition test using anti-Mmc PG-3 antiserum On the basis of these results all the isolates weresuspected to be ofMmc
The PCR amplification in Mmc-specific PCR was foundpositive in all isolates by yielding 195 bp amplicon (Figure 1)the specificity of this PCR was for CAP-21 genomic region[16] However none of the species-specific PCR (mentionedin Table 1) exceptMmc PCRwas amplified against any isolateThus the presence of any other species (M putrefaciens Magalactiae M capricolum subsp capricolum Mmm LC and119872 bovine group 7) was ruled out although they are alsoknown to be associated with goat mastitis milk
Mmc isolates were further studied for any intraspecificstrain variation using 16S ARDRA The 16S rDNA upondigestion withAlu I exhibited strain variation inMmc isolatesby revealing three types of ARDRA patterns (Figure 2) Theisolate numbers 1 2 3 and 4 showed a similar band pattern asthat ofMmc PG-3 by yielding 5 bands (236 186 147 105 and85 bp) while isolate numbers 5 and 6 showed different andunique band patterns by yielding 3 (620 473 and 413 bp) and7 (620 473 413 236 186 147 and 105 bp) bands respectivelywhich were different than the standard strain PG-3 Thesimilarity in band pattern with that of standard strain PG-3 was in agreement with the observations of Stakenborg etal [22] who observed the same pattern for PG-3 using thesame primer and restriction enzyme However the differentband pattern observed in isolates numbers 5 and 6 wasnot in agreement with their observation Our results thatis different band patterns within species are supported byMonnerat et al [19] who also found intraspecific strainvariation in lppA gene ofMmc strains by using Alu I enzymeThe band pattern different from the reference strain (PG-3)observed by us may be attributed to the presence of differentAlu I cutting sites in both of the operons (rrnA and rrnB) asdescribed by Bascunana et al [27]
M 1 2 3 4 5 6
620 bp473 bp413 bp
236 bp186 bp147 bp105 bp85 bp
PG-3
Figure 2 Intra specific strain variation in 16S ARDRA profileamong Mmc isolates M 100 bp DNA ladder PG-3 M mycoidessubsp capri PG-3 Lanes 1ndash6 respective isolates The band thatis 236 bp 186 bp 147 bp and 105 bp are visible very faintly in lanenumber 6
Although M agalactiae is known as the main causativeagent of mastitis [28] along with other species reportedearlier in our case Mmc was isolated from goats havingclinical mastitis as well as from asymptomatic goats Theisolation ofMmc in the present study has also been supportedby the detection of Mmc from milk collected from clinicalmastitis cases in CA endemic area in Spain [9] Our findingsare experimentally supported by Misri et al [2] whoobserved the involvement of Mmc in development of goatmastitis after following the Kochrsquos postulate But the presentfindings are contradictory to earlier reports describing theassociation of goat mastitis with other mycoplasma specieslike M capricolum subsp capricolum M putrefaciens Marginini andMmm LC [6 28ndash31] Since the present study doesnot cover a wide geographic area therefore an isolation workneeds to be carried out at a wider level
In conclusion our finding reports the isolation of Mmchaving intra specific strain variation (in 16S rDNA) fromnatural mastitis in goats which have not been reported everand consequently indicates the association and dually favorsthe earlier report of development of mastitis in goats after theexperimental infection ofMmc Also it reports that in Indiathe occurrence of mycoplasmal mastitis in goats may be duetoMmc infections as no other mycoplasmal species could beisolated from goat mastitis
Conflict of Interests
The authors have no conflict interest to declare
Acknowledgment
The authors are thankful to Dr R A J Nicholas MycoplasmaGroup Veterinary Laboratories Agency (Weybridge) NewHaw Addlestone Surrey UK for providing reference strains
4 ISRN Veterinary Science
of members of mycoides cluster and their monospecificantisera
References
[1] G S Cottew A Breard A J DaMassa et al ldquoTaxonomy of theMycoplasma mycoides clusterrdquo Israel Journal of Medical Sci-ences vol 23 no 6 pp 632ndash635 1987
[2] J Misri P P Gupta and N Sood ldquoExperimental Mycoplasmacapri mastitis in goatsrdquo Australian Veterinary Journal vol 65no 1 pp 33ndash35 1988
[3] R A J Nicholas ldquoImprovements in the diagnosis and controlof diseases of small ruminants caused by mycoplasmasrdquo SmallRuminant Research vol 45 no 2 pp 145ndash149 2002
[4] A Medanet D Zendulkova and Z Pospisil ldquoContagious agal-actia of sheep and goats a reviewrdquo Acta Veterinaria Brno vol70 pp 403ndash412 2001
[5] E O De Azevedo M D B De Alcantara E R Do Nascimentoet al ldquoContagious agalactia by Mycoplasma agalactiae insmall ruminants in Brazil first reportrdquo Brazilian Journal ofMicrobiology vol 37 pp 576ndash581 2006
[6] K Saris ldquoContagious agalactiardquo inMycoplasmas of RuminantsPathogenicity Diagnostics Epidemiology andMolecular GeneticsCOST 826 Agriculture and Biotechnology J Frey and K SarrisEds pp 12ndash15 Luxembourg 1996
[7] H L Ruhnke S Rosendal J Goltz and T E Blackwell ldquoIso-lation of Mycoplasma mycoides subspecies mycoides frompolyarthritis and mastitis of goats in Canadardquo The CanadianVeterinary Journal vol 24 pp 54ndash56 1983
[8] A J DaMassa P S Wakenell and D L Brooks ldquoMycoplasmasof goats and sheeprdquo Journal of Veterinary Diagnostic Investiga-tion vol 4 no 1 pp 101ndash113 1992
[9] J Amores A Sanchez A Gomez-Martın J C Corrales AContreras and C de la Fe ldquoSurveillance of Mycoplasma agal-actiae and Mycoplasma mycoides subsp capri in dairy goatherdsrdquo Small Ruminant Research vol 102 pp 89ndash93 2012
[10] A R DrsquoAngelo A di Provvido G di Francesco et al ldquoExperi-mental infection of goats with an unusual strain ofMycoplasmamycoides subsp Capri isolated in Jordan comparison of differ-ent diagnostic methodsrdquo Veterinaria Italiana vol 46 no 2 pp199ndash207 2010
[11] L E Carmichael T D St George N D Sullivan and NHorsfall ldquoIsolation propagation and characterization studiesof an ovineMycoplasma responsible for proliferative interstitialpneumoniardquo The Cornell Veterinarian vol 62 no 4 pp 654ndash679 1972
[12] W A Clyde ldquoMycoplasma spp Identification based upongrowth inhibition by specific antiserardquo Journal of Immunologyvol 92 no 6 pp 958ndash965 1964
[13] H Ernoslash and L Stipkovits ldquoBovine Mycoplasmas cultural andbiochemical studiesrdquo Acta Veterinaria Scandinavica vol 14 pp436ndash449 1973
[14] F J M Van Kuppeveld J T M Van der Logt A F Angulo et alldquoGenus- and species-specific identification of mycoplasmas by16S rRNA amplificationrdquoApplied and Environmental Microbiol-ogy vol 58 no 8 pp 2606ndash2615 1992
[15] J Sambrook E F Fritsch and T Maniatis Molecular CloningLaboratory Manual Cold Spring Harbor New York NY USA1989
[16] HHotzel K Sachse andH Pfutzner ldquoA PCR scheme for differ-entiation of organisms belonging to the Mycoplasma mycoidesclusterrdquoVeterinaryMicrobiology vol 49 no 1-2 pp 31ndash43 1996
[17] Y R Chavez Gonzalez C R Bascunana G Bolske J G Matts-son C FMolina and K E Johansson ldquoIn vitro amplification ofthe 16S rRNA genes from Mycoplasma bovis and Mycoplasmaagalactiae by PCRrdquo Veterinary Microbiology vol 47 no 1-2 pp183ndash190 1995
[18] A Peyraud S Woubit J B Poveda C De La Fe P Mercier andFThiaucourt ldquoA specific PCR for the detection of Mycoplasmaputrefaciens one of the agents of the contagious agalactiasyndrome of goatsrdquo Molecular and Cellular Probes vol 17 no6 pp 289ndash294 2003
[19] M P Monnerat F Thiaucourt J B Poveda J Nicolet and JFrey ldquoGenetic and serological analysis of lipoprotein LppA inMycoplasma mycoides subsp mycoides LC and Mycoplasmamycoides subsp caprirdquo Clinical and Diagnostic LaboratoryImmunology vol 6 no 2 pp 224ndash230 1999
[20] J Frey X Cheng M P Monnerat et al ldquoGenetic and sero-logical analysis of the immunogenic 67-kDa lipoprotein ofMycoplasma sp Bovine group 7rdquo Research in Microbiology vol149 no 1 pp 55ndash64 1998
[21] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989
[22] T Stakenborg J Vicca R Verhelst et al ldquoEvaluation of tRNAgene PCR for identification of mollicutesrdquo Journal of ClinicalMicrobiology vol 43 no 9 pp 4558ndash4566 2005
[23] S Razin and E A Freundt ldquoThe Mollicutes Mycoplasmatalesand Mycoplasmatacaerdquo in Bergeyrsquos Manual of Systematic Bacte-riology N R Krieg and J G Holt Eds vol 1 pp 740ndash742 TheWilliams ampWilkins Baltimore Md USA 1984
[24] T D V M Sori A D V M Zeleke E D V M Gelaye and F DV M Regassa ldquoIsolation and identification of Mycoplasmamycoides subsp mycoides small colony type in Eastern Ethi-opiardquo International Journal of Applied Research in VeterinaryMedicine vol 3 no 1 pp 30ndash34 2005
[25] J O Ikheloa A T P Ajuwape and A I Adetosoye ldquoBiochemi-cal characterization and serological identification of mycoplas-mas isolated from pneumonic lungs of goats slaughtered inabattoirs in Northern Nigeriardquo Small Ruminant Research vol52 no 1-2 pp 93ndash97 2004
[26] M C Gil M De Hermoso Mendoza J Rey J M Alonso J BPoveda and J De Hermoso Mendoza ldquoAetiology of caprinecontagious agalactia syndrome in Extremadura Spainrdquo Veteri-nary Record vol 144 no 1 pp 24ndash25 1999
[27] C R Bascunana J GMattsson G Bolske and K E JohanssonldquoCharacterization of the 16S rRNA genes from Mycoplasma spstrain F38 and development of an identification system basedon PCRrdquo Journal of Bacteriology vol 176 no 9 pp 2577ndash25861994
[28] D Bergonier X Berthelot and F Poumarat ldquoContagious agal-actia of small ruminants current knowledge concerning epi-demiology diagnosis and controlrdquo OIE Revue Scientifique etTechnique vol 16 no 3 pp 848ndash873 1997
[29] Bar-Moshe E Rapport E Bogin and E Lebel ldquoVaccinationtrial against caprine Mycoplasma mycoids subspmycoids (LCtype) infection in goats infectivity trials vaccination andchallengerdquo Refuah Veterinarith vol 39 pp 77ndash88 1982
ISRN Veterinary Science 5
[30] A J DaMassa D L Brooks C A Holmberg and A I MoeldquoCaprine mycoplasmosis an outbreak of mastitis and arthritisrequiring the destruction of 700 goatsrdquo Veterinary Record vol120 no 17 pp 409ndash413 1987
[31] P Kumar A Roy B B Bhanderi and B C Pal ldquoIsolationlsquoiden-tification and molecular characterization of Mycoplasma iso-lates from goats of Gujarat State Indiardquo Veterinarski Arhiv vol81 no 4 pp 443ndash458 2011
lowastMmcM mycoides subsp capriMmm LCM mycoides susp mycoides large colony typeM agM agalactiaeMccM capricolum subsp capricolumMbg7M bovine group 7
for sampling Goats not exhibiting clinical mastitis weretaken as asymptomatic ones and suspected for carriers ofmycoplasmas
22 Goat Herd Sample Collection and Isolation of Mycoplas-mas A total of 171 goats were sampled for milk from fivedifferent goat herds of Mathura region (CIRG MakhdoomJhandipur Chattar Singh Ka Nagla Keetham and Agra)that facing the problems of CA andor mastitis Out of 171milk samples 102 were from clinical mastitis and 69 fromasymptomatic goats Milk samples were collected asepticallyin sterile vials that is before milking affected teats werecleaned with 70 ethanol and then the first 2-3 streams werediscarded
Isolation of mycoplasmas from milk samples was per-formed as described by Carmichael et al [11] with slightmodifications Briefly milk samples were inoculated intoliquid Hankrsquos balanced salt solution (HBSS-L pH 76ndash78)medium and incubated at 37∘C under 5 CO
2for up to 14
days and subsequently transferred on solid Hankrsquos balancedsalt solution (HBSS-S) medium The probability of L phasevariants was ruled out by forward and reversal passages inthe artificial medium
23 Biochemical Characterization A preliminary characteri-zation of the isolates was performed by digitonin sensitivityand growth inhibition tests as per the method describedelsewhere [12] and Giemsa method of staining This wasfollowed by biochemical tests namely glucose fermentationphosphate reduction gelatin hydrolysis and film and spotformation test as described earlier [13]
24 Confirmation of Isolates by PCR Genomic DNA ofthe isolates was extracted from late exponential growthphase using the phenol-chloroformmethod described by vanKuppeveled et al [14] and purity and concentration of DNAwas checked on 07 agarose gel and spectrophotometricanalysis according to Sambrook et al [15]
Mycoplasma isolates were confirmed by employingMmc-specific PCR and the presence of other mycoplasmas wasruled out by conducting the respective species-specific PCRsaccording to respective protocols described elsewhere asreferred in Table 1 The Qiagen PCR core kit was used toperform all the PCRs and consequent PCR products werechecked on 2 agarose gel
25 Characterization of Intraspecies Strain Variation Using 16SARDRA The 16S rDNA of all isolates was amplified by usingthe universal primer pair pA (51015840-AGAGTTTGATCCTGG-CTCAG-31015840) and pH (51015840-AAGGAGGTGATCCAGCCGCA-31015840) for 30 cycles (20 sec 94∘C 15 sec 57∘C and 30 sec72∘C) using Qiagen PCR core kit as per Edwards et al[21] The resultant amplicon (1500 bp) was purified by usingpurification kit (Bangalore Genei India) It was subsequentlydigested with restriction enzymeAlu I (Fermentas sequenceAGandCT) and the restriction fragments were separated on 3NuSieve 3 1 agar by using the method of Stakenborg et al[22]
3 Results and Discussion
Out of 171 clinical and asymptomatic samples a total of45 samples showed fine turbidity and pH shift (acidic)imparting a yellow color to the broth medium within 3 to10 days indicating the mycoplasma growth After followingthe protocol of 4-5 reversal and 3-4 forward passages thepossibility of ldquoL phase variantrdquo was ruled out Only 6 (35)samples yielded colonies of 1 to 2mm size exhibiting typicalfried egg appearance on HBSS-S medium Their growthcharacteristics were indicative of the mycoplasmas Of sixisolates 5 were recovered from clinical mastitis milk whereasone (isolate number 6) was from subclinical mastitis milkThese growth evidences were in accordance with Razin andFreundt [23] and Sori et al [24] In the study the isolationrate (35) was found to be in agreement with Ikhloea et al[25] who obtained similar results of 37 to 11 however our
ISRN Veterinary Science 3
PC 1 NC 2 3 4 5 6
M
Figure 1 M mycoides subsp Capri-specific PCR exhibiting 195 bpamplicon M 100 bp DNA ladder PC positive control (M mycoidessubsp capri PG-3) NC negative control Lanes 1ndash6 respectiveisolates
isolation rate seems to be quite low in contrast to the 25 to71 obtained by Gil et al [26]
All the isolates showed purplish-pinkish coccobacillarybodies with pleomorphic shape and size upon Giemsa stain-ing The isolates passed filtration test through 045120583m filterand found to be sensitive to digitonin Biochemical testsnamely glucose fermentation and gelatin hydrolysis testsgave positive results while film and spot formation test andphosphatase test were negative for all isolates The isolatesexhibited positive growth inhibition test using anti-Mmc PG-3 antiserum On the basis of these results all the isolates weresuspected to be ofMmc
The PCR amplification in Mmc-specific PCR was foundpositive in all isolates by yielding 195 bp amplicon (Figure 1)the specificity of this PCR was for CAP-21 genomic region[16] However none of the species-specific PCR (mentionedin Table 1) exceptMmc PCRwas amplified against any isolateThus the presence of any other species (M putrefaciens Magalactiae M capricolum subsp capricolum Mmm LC and119872 bovine group 7) was ruled out although they are alsoknown to be associated with goat mastitis milk
Mmc isolates were further studied for any intraspecificstrain variation using 16S ARDRA The 16S rDNA upondigestion withAlu I exhibited strain variation inMmc isolatesby revealing three types of ARDRA patterns (Figure 2) Theisolate numbers 1 2 3 and 4 showed a similar band pattern asthat ofMmc PG-3 by yielding 5 bands (236 186 147 105 and85 bp) while isolate numbers 5 and 6 showed different andunique band patterns by yielding 3 (620 473 and 413 bp) and7 (620 473 413 236 186 147 and 105 bp) bands respectivelywhich were different than the standard strain PG-3 Thesimilarity in band pattern with that of standard strain PG-3 was in agreement with the observations of Stakenborg etal [22] who observed the same pattern for PG-3 using thesame primer and restriction enzyme However the differentband pattern observed in isolates numbers 5 and 6 wasnot in agreement with their observation Our results thatis different band patterns within species are supported byMonnerat et al [19] who also found intraspecific strainvariation in lppA gene ofMmc strains by using Alu I enzymeThe band pattern different from the reference strain (PG-3)observed by us may be attributed to the presence of differentAlu I cutting sites in both of the operons (rrnA and rrnB) asdescribed by Bascunana et al [27]
M 1 2 3 4 5 6
620 bp473 bp413 bp
236 bp186 bp147 bp105 bp85 bp
PG-3
Figure 2 Intra specific strain variation in 16S ARDRA profileamong Mmc isolates M 100 bp DNA ladder PG-3 M mycoidessubsp capri PG-3 Lanes 1ndash6 respective isolates The band thatis 236 bp 186 bp 147 bp and 105 bp are visible very faintly in lanenumber 6
Although M agalactiae is known as the main causativeagent of mastitis [28] along with other species reportedearlier in our case Mmc was isolated from goats havingclinical mastitis as well as from asymptomatic goats Theisolation ofMmc in the present study has also been supportedby the detection of Mmc from milk collected from clinicalmastitis cases in CA endemic area in Spain [9] Our findingsare experimentally supported by Misri et al [2] whoobserved the involvement of Mmc in development of goatmastitis after following the Kochrsquos postulate But the presentfindings are contradictory to earlier reports describing theassociation of goat mastitis with other mycoplasma specieslike M capricolum subsp capricolum M putrefaciens Marginini andMmm LC [6 28ndash31] Since the present study doesnot cover a wide geographic area therefore an isolation workneeds to be carried out at a wider level
In conclusion our finding reports the isolation of Mmchaving intra specific strain variation (in 16S rDNA) fromnatural mastitis in goats which have not been reported everand consequently indicates the association and dually favorsthe earlier report of development of mastitis in goats after theexperimental infection ofMmc Also it reports that in Indiathe occurrence of mycoplasmal mastitis in goats may be duetoMmc infections as no other mycoplasmal species could beisolated from goat mastitis
Conflict of Interests
The authors have no conflict interest to declare
Acknowledgment
The authors are thankful to Dr R A J Nicholas MycoplasmaGroup Veterinary Laboratories Agency (Weybridge) NewHaw Addlestone Surrey UK for providing reference strains
4 ISRN Veterinary Science
of members of mycoides cluster and their monospecificantisera
References
[1] G S Cottew A Breard A J DaMassa et al ldquoTaxonomy of theMycoplasma mycoides clusterrdquo Israel Journal of Medical Sci-ences vol 23 no 6 pp 632ndash635 1987
[2] J Misri P P Gupta and N Sood ldquoExperimental Mycoplasmacapri mastitis in goatsrdquo Australian Veterinary Journal vol 65no 1 pp 33ndash35 1988
[3] R A J Nicholas ldquoImprovements in the diagnosis and controlof diseases of small ruminants caused by mycoplasmasrdquo SmallRuminant Research vol 45 no 2 pp 145ndash149 2002
[4] A Medanet D Zendulkova and Z Pospisil ldquoContagious agal-actia of sheep and goats a reviewrdquo Acta Veterinaria Brno vol70 pp 403ndash412 2001
[5] E O De Azevedo M D B De Alcantara E R Do Nascimentoet al ldquoContagious agalactia by Mycoplasma agalactiae insmall ruminants in Brazil first reportrdquo Brazilian Journal ofMicrobiology vol 37 pp 576ndash581 2006
[6] K Saris ldquoContagious agalactiardquo inMycoplasmas of RuminantsPathogenicity Diagnostics Epidemiology andMolecular GeneticsCOST 826 Agriculture and Biotechnology J Frey and K SarrisEds pp 12ndash15 Luxembourg 1996
[7] H L Ruhnke S Rosendal J Goltz and T E Blackwell ldquoIso-lation of Mycoplasma mycoides subspecies mycoides frompolyarthritis and mastitis of goats in Canadardquo The CanadianVeterinary Journal vol 24 pp 54ndash56 1983
[8] A J DaMassa P S Wakenell and D L Brooks ldquoMycoplasmasof goats and sheeprdquo Journal of Veterinary Diagnostic Investiga-tion vol 4 no 1 pp 101ndash113 1992
[9] J Amores A Sanchez A Gomez-Martın J C Corrales AContreras and C de la Fe ldquoSurveillance of Mycoplasma agal-actiae and Mycoplasma mycoides subsp capri in dairy goatherdsrdquo Small Ruminant Research vol 102 pp 89ndash93 2012
[10] A R DrsquoAngelo A di Provvido G di Francesco et al ldquoExperi-mental infection of goats with an unusual strain ofMycoplasmamycoides subsp Capri isolated in Jordan comparison of differ-ent diagnostic methodsrdquo Veterinaria Italiana vol 46 no 2 pp199ndash207 2010
[11] L E Carmichael T D St George N D Sullivan and NHorsfall ldquoIsolation propagation and characterization studiesof an ovineMycoplasma responsible for proliferative interstitialpneumoniardquo The Cornell Veterinarian vol 62 no 4 pp 654ndash679 1972
[12] W A Clyde ldquoMycoplasma spp Identification based upongrowth inhibition by specific antiserardquo Journal of Immunologyvol 92 no 6 pp 958ndash965 1964
[13] H Ernoslash and L Stipkovits ldquoBovine Mycoplasmas cultural andbiochemical studiesrdquo Acta Veterinaria Scandinavica vol 14 pp436ndash449 1973
[14] F J M Van Kuppeveld J T M Van der Logt A F Angulo et alldquoGenus- and species-specific identification of mycoplasmas by16S rRNA amplificationrdquoApplied and Environmental Microbiol-ogy vol 58 no 8 pp 2606ndash2615 1992
[15] J Sambrook E F Fritsch and T Maniatis Molecular CloningLaboratory Manual Cold Spring Harbor New York NY USA1989
[16] HHotzel K Sachse andH Pfutzner ldquoA PCR scheme for differ-entiation of organisms belonging to the Mycoplasma mycoidesclusterrdquoVeterinaryMicrobiology vol 49 no 1-2 pp 31ndash43 1996
[17] Y R Chavez Gonzalez C R Bascunana G Bolske J G Matts-son C FMolina and K E Johansson ldquoIn vitro amplification ofthe 16S rRNA genes from Mycoplasma bovis and Mycoplasmaagalactiae by PCRrdquo Veterinary Microbiology vol 47 no 1-2 pp183ndash190 1995
[18] A Peyraud S Woubit J B Poveda C De La Fe P Mercier andFThiaucourt ldquoA specific PCR for the detection of Mycoplasmaputrefaciens one of the agents of the contagious agalactiasyndrome of goatsrdquo Molecular and Cellular Probes vol 17 no6 pp 289ndash294 2003
[19] M P Monnerat F Thiaucourt J B Poveda J Nicolet and JFrey ldquoGenetic and serological analysis of lipoprotein LppA inMycoplasma mycoides subsp mycoides LC and Mycoplasmamycoides subsp caprirdquo Clinical and Diagnostic LaboratoryImmunology vol 6 no 2 pp 224ndash230 1999
[20] J Frey X Cheng M P Monnerat et al ldquoGenetic and sero-logical analysis of the immunogenic 67-kDa lipoprotein ofMycoplasma sp Bovine group 7rdquo Research in Microbiology vol149 no 1 pp 55ndash64 1998
[21] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989
[22] T Stakenborg J Vicca R Verhelst et al ldquoEvaluation of tRNAgene PCR for identification of mollicutesrdquo Journal of ClinicalMicrobiology vol 43 no 9 pp 4558ndash4566 2005
[23] S Razin and E A Freundt ldquoThe Mollicutes Mycoplasmatalesand Mycoplasmatacaerdquo in Bergeyrsquos Manual of Systematic Bacte-riology N R Krieg and J G Holt Eds vol 1 pp 740ndash742 TheWilliams ampWilkins Baltimore Md USA 1984
[24] T D V M Sori A D V M Zeleke E D V M Gelaye and F DV M Regassa ldquoIsolation and identification of Mycoplasmamycoides subsp mycoides small colony type in Eastern Ethi-opiardquo International Journal of Applied Research in VeterinaryMedicine vol 3 no 1 pp 30ndash34 2005
[25] J O Ikheloa A T P Ajuwape and A I Adetosoye ldquoBiochemi-cal characterization and serological identification of mycoplas-mas isolated from pneumonic lungs of goats slaughtered inabattoirs in Northern Nigeriardquo Small Ruminant Research vol52 no 1-2 pp 93ndash97 2004
[26] M C Gil M De Hermoso Mendoza J Rey J M Alonso J BPoveda and J De Hermoso Mendoza ldquoAetiology of caprinecontagious agalactia syndrome in Extremadura Spainrdquo Veteri-nary Record vol 144 no 1 pp 24ndash25 1999
[27] C R Bascunana J GMattsson G Bolske and K E JohanssonldquoCharacterization of the 16S rRNA genes from Mycoplasma spstrain F38 and development of an identification system basedon PCRrdquo Journal of Bacteriology vol 176 no 9 pp 2577ndash25861994
[28] D Bergonier X Berthelot and F Poumarat ldquoContagious agal-actia of small ruminants current knowledge concerning epi-demiology diagnosis and controlrdquo OIE Revue Scientifique etTechnique vol 16 no 3 pp 848ndash873 1997
[29] Bar-Moshe E Rapport E Bogin and E Lebel ldquoVaccinationtrial against caprine Mycoplasma mycoids subspmycoids (LCtype) infection in goats infectivity trials vaccination andchallengerdquo Refuah Veterinarith vol 39 pp 77ndash88 1982
ISRN Veterinary Science 5
[30] A J DaMassa D L Brooks C A Holmberg and A I MoeldquoCaprine mycoplasmosis an outbreak of mastitis and arthritisrequiring the destruction of 700 goatsrdquo Veterinary Record vol120 no 17 pp 409ndash413 1987
[31] P Kumar A Roy B B Bhanderi and B C Pal ldquoIsolationlsquoiden-tification and molecular characterization of Mycoplasma iso-lates from goats of Gujarat State Indiardquo Veterinarski Arhiv vol81 no 4 pp 443ndash458 2011
Figure 1 M mycoides subsp Capri-specific PCR exhibiting 195 bpamplicon M 100 bp DNA ladder PC positive control (M mycoidessubsp capri PG-3) NC negative control Lanes 1ndash6 respectiveisolates
isolation rate seems to be quite low in contrast to the 25 to71 obtained by Gil et al [26]
All the isolates showed purplish-pinkish coccobacillarybodies with pleomorphic shape and size upon Giemsa stain-ing The isolates passed filtration test through 045120583m filterand found to be sensitive to digitonin Biochemical testsnamely glucose fermentation and gelatin hydrolysis testsgave positive results while film and spot formation test andphosphatase test were negative for all isolates The isolatesexhibited positive growth inhibition test using anti-Mmc PG-3 antiserum On the basis of these results all the isolates weresuspected to be ofMmc
The PCR amplification in Mmc-specific PCR was foundpositive in all isolates by yielding 195 bp amplicon (Figure 1)the specificity of this PCR was for CAP-21 genomic region[16] However none of the species-specific PCR (mentionedin Table 1) exceptMmc PCRwas amplified against any isolateThus the presence of any other species (M putrefaciens Magalactiae M capricolum subsp capricolum Mmm LC and119872 bovine group 7) was ruled out although they are alsoknown to be associated with goat mastitis milk
Mmc isolates were further studied for any intraspecificstrain variation using 16S ARDRA The 16S rDNA upondigestion withAlu I exhibited strain variation inMmc isolatesby revealing three types of ARDRA patterns (Figure 2) Theisolate numbers 1 2 3 and 4 showed a similar band pattern asthat ofMmc PG-3 by yielding 5 bands (236 186 147 105 and85 bp) while isolate numbers 5 and 6 showed different andunique band patterns by yielding 3 (620 473 and 413 bp) and7 (620 473 413 236 186 147 and 105 bp) bands respectivelywhich were different than the standard strain PG-3 Thesimilarity in band pattern with that of standard strain PG-3 was in agreement with the observations of Stakenborg etal [22] who observed the same pattern for PG-3 using thesame primer and restriction enzyme However the differentband pattern observed in isolates numbers 5 and 6 wasnot in agreement with their observation Our results thatis different band patterns within species are supported byMonnerat et al [19] who also found intraspecific strainvariation in lppA gene ofMmc strains by using Alu I enzymeThe band pattern different from the reference strain (PG-3)observed by us may be attributed to the presence of differentAlu I cutting sites in both of the operons (rrnA and rrnB) asdescribed by Bascunana et al [27]
M 1 2 3 4 5 6
620 bp473 bp413 bp
236 bp186 bp147 bp105 bp85 bp
PG-3
Figure 2 Intra specific strain variation in 16S ARDRA profileamong Mmc isolates M 100 bp DNA ladder PG-3 M mycoidessubsp capri PG-3 Lanes 1ndash6 respective isolates The band thatis 236 bp 186 bp 147 bp and 105 bp are visible very faintly in lanenumber 6
Although M agalactiae is known as the main causativeagent of mastitis [28] along with other species reportedearlier in our case Mmc was isolated from goats havingclinical mastitis as well as from asymptomatic goats Theisolation ofMmc in the present study has also been supportedby the detection of Mmc from milk collected from clinicalmastitis cases in CA endemic area in Spain [9] Our findingsare experimentally supported by Misri et al [2] whoobserved the involvement of Mmc in development of goatmastitis after following the Kochrsquos postulate But the presentfindings are contradictory to earlier reports describing theassociation of goat mastitis with other mycoplasma specieslike M capricolum subsp capricolum M putrefaciens Marginini andMmm LC [6 28ndash31] Since the present study doesnot cover a wide geographic area therefore an isolation workneeds to be carried out at a wider level
In conclusion our finding reports the isolation of Mmchaving intra specific strain variation (in 16S rDNA) fromnatural mastitis in goats which have not been reported everand consequently indicates the association and dually favorsthe earlier report of development of mastitis in goats after theexperimental infection ofMmc Also it reports that in Indiathe occurrence of mycoplasmal mastitis in goats may be duetoMmc infections as no other mycoplasmal species could beisolated from goat mastitis
Conflict of Interests
The authors have no conflict interest to declare
Acknowledgment
The authors are thankful to Dr R A J Nicholas MycoplasmaGroup Veterinary Laboratories Agency (Weybridge) NewHaw Addlestone Surrey UK for providing reference strains
4 ISRN Veterinary Science
of members of mycoides cluster and their monospecificantisera
References
[1] G S Cottew A Breard A J DaMassa et al ldquoTaxonomy of theMycoplasma mycoides clusterrdquo Israel Journal of Medical Sci-ences vol 23 no 6 pp 632ndash635 1987
[2] J Misri P P Gupta and N Sood ldquoExperimental Mycoplasmacapri mastitis in goatsrdquo Australian Veterinary Journal vol 65no 1 pp 33ndash35 1988
[3] R A J Nicholas ldquoImprovements in the diagnosis and controlof diseases of small ruminants caused by mycoplasmasrdquo SmallRuminant Research vol 45 no 2 pp 145ndash149 2002
[4] A Medanet D Zendulkova and Z Pospisil ldquoContagious agal-actia of sheep and goats a reviewrdquo Acta Veterinaria Brno vol70 pp 403ndash412 2001
[5] E O De Azevedo M D B De Alcantara E R Do Nascimentoet al ldquoContagious agalactia by Mycoplasma agalactiae insmall ruminants in Brazil first reportrdquo Brazilian Journal ofMicrobiology vol 37 pp 576ndash581 2006
[6] K Saris ldquoContagious agalactiardquo inMycoplasmas of RuminantsPathogenicity Diagnostics Epidemiology andMolecular GeneticsCOST 826 Agriculture and Biotechnology J Frey and K SarrisEds pp 12ndash15 Luxembourg 1996
[7] H L Ruhnke S Rosendal J Goltz and T E Blackwell ldquoIso-lation of Mycoplasma mycoides subspecies mycoides frompolyarthritis and mastitis of goats in Canadardquo The CanadianVeterinary Journal vol 24 pp 54ndash56 1983
[8] A J DaMassa P S Wakenell and D L Brooks ldquoMycoplasmasof goats and sheeprdquo Journal of Veterinary Diagnostic Investiga-tion vol 4 no 1 pp 101ndash113 1992
[9] J Amores A Sanchez A Gomez-Martın J C Corrales AContreras and C de la Fe ldquoSurveillance of Mycoplasma agal-actiae and Mycoplasma mycoides subsp capri in dairy goatherdsrdquo Small Ruminant Research vol 102 pp 89ndash93 2012
[10] A R DrsquoAngelo A di Provvido G di Francesco et al ldquoExperi-mental infection of goats with an unusual strain ofMycoplasmamycoides subsp Capri isolated in Jordan comparison of differ-ent diagnostic methodsrdquo Veterinaria Italiana vol 46 no 2 pp199ndash207 2010
[11] L E Carmichael T D St George N D Sullivan and NHorsfall ldquoIsolation propagation and characterization studiesof an ovineMycoplasma responsible for proliferative interstitialpneumoniardquo The Cornell Veterinarian vol 62 no 4 pp 654ndash679 1972
[12] W A Clyde ldquoMycoplasma spp Identification based upongrowth inhibition by specific antiserardquo Journal of Immunologyvol 92 no 6 pp 958ndash965 1964
[13] H Ernoslash and L Stipkovits ldquoBovine Mycoplasmas cultural andbiochemical studiesrdquo Acta Veterinaria Scandinavica vol 14 pp436ndash449 1973
[14] F J M Van Kuppeveld J T M Van der Logt A F Angulo et alldquoGenus- and species-specific identification of mycoplasmas by16S rRNA amplificationrdquoApplied and Environmental Microbiol-ogy vol 58 no 8 pp 2606ndash2615 1992
[15] J Sambrook E F Fritsch and T Maniatis Molecular CloningLaboratory Manual Cold Spring Harbor New York NY USA1989
[16] HHotzel K Sachse andH Pfutzner ldquoA PCR scheme for differ-entiation of organisms belonging to the Mycoplasma mycoidesclusterrdquoVeterinaryMicrobiology vol 49 no 1-2 pp 31ndash43 1996
[17] Y R Chavez Gonzalez C R Bascunana G Bolske J G Matts-son C FMolina and K E Johansson ldquoIn vitro amplification ofthe 16S rRNA genes from Mycoplasma bovis and Mycoplasmaagalactiae by PCRrdquo Veterinary Microbiology vol 47 no 1-2 pp183ndash190 1995
[18] A Peyraud S Woubit J B Poveda C De La Fe P Mercier andFThiaucourt ldquoA specific PCR for the detection of Mycoplasmaputrefaciens one of the agents of the contagious agalactiasyndrome of goatsrdquo Molecular and Cellular Probes vol 17 no6 pp 289ndash294 2003
[19] M P Monnerat F Thiaucourt J B Poveda J Nicolet and JFrey ldquoGenetic and serological analysis of lipoprotein LppA inMycoplasma mycoides subsp mycoides LC and Mycoplasmamycoides subsp caprirdquo Clinical and Diagnostic LaboratoryImmunology vol 6 no 2 pp 224ndash230 1999
[20] J Frey X Cheng M P Monnerat et al ldquoGenetic and sero-logical analysis of the immunogenic 67-kDa lipoprotein ofMycoplasma sp Bovine group 7rdquo Research in Microbiology vol149 no 1 pp 55ndash64 1998
[21] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989
[22] T Stakenborg J Vicca R Verhelst et al ldquoEvaluation of tRNAgene PCR for identification of mollicutesrdquo Journal of ClinicalMicrobiology vol 43 no 9 pp 4558ndash4566 2005
[23] S Razin and E A Freundt ldquoThe Mollicutes Mycoplasmatalesand Mycoplasmatacaerdquo in Bergeyrsquos Manual of Systematic Bacte-riology N R Krieg and J G Holt Eds vol 1 pp 740ndash742 TheWilliams ampWilkins Baltimore Md USA 1984
[24] T D V M Sori A D V M Zeleke E D V M Gelaye and F DV M Regassa ldquoIsolation and identification of Mycoplasmamycoides subsp mycoides small colony type in Eastern Ethi-opiardquo International Journal of Applied Research in VeterinaryMedicine vol 3 no 1 pp 30ndash34 2005
[25] J O Ikheloa A T P Ajuwape and A I Adetosoye ldquoBiochemi-cal characterization and serological identification of mycoplas-mas isolated from pneumonic lungs of goats slaughtered inabattoirs in Northern Nigeriardquo Small Ruminant Research vol52 no 1-2 pp 93ndash97 2004
[26] M C Gil M De Hermoso Mendoza J Rey J M Alonso J BPoveda and J De Hermoso Mendoza ldquoAetiology of caprinecontagious agalactia syndrome in Extremadura Spainrdquo Veteri-nary Record vol 144 no 1 pp 24ndash25 1999
[27] C R Bascunana J GMattsson G Bolske and K E JohanssonldquoCharacterization of the 16S rRNA genes from Mycoplasma spstrain F38 and development of an identification system basedon PCRrdquo Journal of Bacteriology vol 176 no 9 pp 2577ndash25861994
[28] D Bergonier X Berthelot and F Poumarat ldquoContagious agal-actia of small ruminants current knowledge concerning epi-demiology diagnosis and controlrdquo OIE Revue Scientifique etTechnique vol 16 no 3 pp 848ndash873 1997
[29] Bar-Moshe E Rapport E Bogin and E Lebel ldquoVaccinationtrial against caprine Mycoplasma mycoids subspmycoids (LCtype) infection in goats infectivity trials vaccination andchallengerdquo Refuah Veterinarith vol 39 pp 77ndash88 1982
ISRN Veterinary Science 5
[30] A J DaMassa D L Brooks C A Holmberg and A I MoeldquoCaprine mycoplasmosis an outbreak of mastitis and arthritisrequiring the destruction of 700 goatsrdquo Veterinary Record vol120 no 17 pp 409ndash413 1987
[31] P Kumar A Roy B B Bhanderi and B C Pal ldquoIsolationlsquoiden-tification and molecular characterization of Mycoplasma iso-lates from goats of Gujarat State Indiardquo Veterinarski Arhiv vol81 no 4 pp 443ndash458 2011
of members of mycoides cluster and their monospecificantisera
References
[1] G S Cottew A Breard A J DaMassa et al ldquoTaxonomy of theMycoplasma mycoides clusterrdquo Israel Journal of Medical Sci-ences vol 23 no 6 pp 632ndash635 1987
[2] J Misri P P Gupta and N Sood ldquoExperimental Mycoplasmacapri mastitis in goatsrdquo Australian Veterinary Journal vol 65no 1 pp 33ndash35 1988
[3] R A J Nicholas ldquoImprovements in the diagnosis and controlof diseases of small ruminants caused by mycoplasmasrdquo SmallRuminant Research vol 45 no 2 pp 145ndash149 2002
[4] A Medanet D Zendulkova and Z Pospisil ldquoContagious agal-actia of sheep and goats a reviewrdquo Acta Veterinaria Brno vol70 pp 403ndash412 2001
[5] E O De Azevedo M D B De Alcantara E R Do Nascimentoet al ldquoContagious agalactia by Mycoplasma agalactiae insmall ruminants in Brazil first reportrdquo Brazilian Journal ofMicrobiology vol 37 pp 576ndash581 2006
[6] K Saris ldquoContagious agalactiardquo inMycoplasmas of RuminantsPathogenicity Diagnostics Epidemiology andMolecular GeneticsCOST 826 Agriculture and Biotechnology J Frey and K SarrisEds pp 12ndash15 Luxembourg 1996
[7] H L Ruhnke S Rosendal J Goltz and T E Blackwell ldquoIso-lation of Mycoplasma mycoides subspecies mycoides frompolyarthritis and mastitis of goats in Canadardquo The CanadianVeterinary Journal vol 24 pp 54ndash56 1983
[8] A J DaMassa P S Wakenell and D L Brooks ldquoMycoplasmasof goats and sheeprdquo Journal of Veterinary Diagnostic Investiga-tion vol 4 no 1 pp 101ndash113 1992
[9] J Amores A Sanchez A Gomez-Martın J C Corrales AContreras and C de la Fe ldquoSurveillance of Mycoplasma agal-actiae and Mycoplasma mycoides subsp capri in dairy goatherdsrdquo Small Ruminant Research vol 102 pp 89ndash93 2012
[10] A R DrsquoAngelo A di Provvido G di Francesco et al ldquoExperi-mental infection of goats with an unusual strain ofMycoplasmamycoides subsp Capri isolated in Jordan comparison of differ-ent diagnostic methodsrdquo Veterinaria Italiana vol 46 no 2 pp199ndash207 2010
[11] L E Carmichael T D St George N D Sullivan and NHorsfall ldquoIsolation propagation and characterization studiesof an ovineMycoplasma responsible for proliferative interstitialpneumoniardquo The Cornell Veterinarian vol 62 no 4 pp 654ndash679 1972
[12] W A Clyde ldquoMycoplasma spp Identification based upongrowth inhibition by specific antiserardquo Journal of Immunologyvol 92 no 6 pp 958ndash965 1964
[13] H Ernoslash and L Stipkovits ldquoBovine Mycoplasmas cultural andbiochemical studiesrdquo Acta Veterinaria Scandinavica vol 14 pp436ndash449 1973
[14] F J M Van Kuppeveld J T M Van der Logt A F Angulo et alldquoGenus- and species-specific identification of mycoplasmas by16S rRNA amplificationrdquoApplied and Environmental Microbiol-ogy vol 58 no 8 pp 2606ndash2615 1992
[15] J Sambrook E F Fritsch and T Maniatis Molecular CloningLaboratory Manual Cold Spring Harbor New York NY USA1989
[16] HHotzel K Sachse andH Pfutzner ldquoA PCR scheme for differ-entiation of organisms belonging to the Mycoplasma mycoidesclusterrdquoVeterinaryMicrobiology vol 49 no 1-2 pp 31ndash43 1996
[17] Y R Chavez Gonzalez C R Bascunana G Bolske J G Matts-son C FMolina and K E Johansson ldquoIn vitro amplification ofthe 16S rRNA genes from Mycoplasma bovis and Mycoplasmaagalactiae by PCRrdquo Veterinary Microbiology vol 47 no 1-2 pp183ndash190 1995
[18] A Peyraud S Woubit J B Poveda C De La Fe P Mercier andFThiaucourt ldquoA specific PCR for the detection of Mycoplasmaputrefaciens one of the agents of the contagious agalactiasyndrome of goatsrdquo Molecular and Cellular Probes vol 17 no6 pp 289ndash294 2003
[19] M P Monnerat F Thiaucourt J B Poveda J Nicolet and JFrey ldquoGenetic and serological analysis of lipoprotein LppA inMycoplasma mycoides subsp mycoides LC and Mycoplasmamycoides subsp caprirdquo Clinical and Diagnostic LaboratoryImmunology vol 6 no 2 pp 224ndash230 1999
[20] J Frey X Cheng M P Monnerat et al ldquoGenetic and sero-logical analysis of the immunogenic 67-kDa lipoprotein ofMycoplasma sp Bovine group 7rdquo Research in Microbiology vol149 no 1 pp 55ndash64 1998
[21] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989
[22] T Stakenborg J Vicca R Verhelst et al ldquoEvaluation of tRNAgene PCR for identification of mollicutesrdquo Journal of ClinicalMicrobiology vol 43 no 9 pp 4558ndash4566 2005
[23] S Razin and E A Freundt ldquoThe Mollicutes Mycoplasmatalesand Mycoplasmatacaerdquo in Bergeyrsquos Manual of Systematic Bacte-riology N R Krieg and J G Holt Eds vol 1 pp 740ndash742 TheWilliams ampWilkins Baltimore Md USA 1984
[24] T D V M Sori A D V M Zeleke E D V M Gelaye and F DV M Regassa ldquoIsolation and identification of Mycoplasmamycoides subsp mycoides small colony type in Eastern Ethi-opiardquo International Journal of Applied Research in VeterinaryMedicine vol 3 no 1 pp 30ndash34 2005
[25] J O Ikheloa A T P Ajuwape and A I Adetosoye ldquoBiochemi-cal characterization and serological identification of mycoplas-mas isolated from pneumonic lungs of goats slaughtered inabattoirs in Northern Nigeriardquo Small Ruminant Research vol52 no 1-2 pp 93ndash97 2004
[26] M C Gil M De Hermoso Mendoza J Rey J M Alonso J BPoveda and J De Hermoso Mendoza ldquoAetiology of caprinecontagious agalactia syndrome in Extremadura Spainrdquo Veteri-nary Record vol 144 no 1 pp 24ndash25 1999
[27] C R Bascunana J GMattsson G Bolske and K E JohanssonldquoCharacterization of the 16S rRNA genes from Mycoplasma spstrain F38 and development of an identification system basedon PCRrdquo Journal of Bacteriology vol 176 no 9 pp 2577ndash25861994
[28] D Bergonier X Berthelot and F Poumarat ldquoContagious agal-actia of small ruminants current knowledge concerning epi-demiology diagnosis and controlrdquo OIE Revue Scientifique etTechnique vol 16 no 3 pp 848ndash873 1997
[29] Bar-Moshe E Rapport E Bogin and E Lebel ldquoVaccinationtrial against caprine Mycoplasma mycoids subspmycoids (LCtype) infection in goats infectivity trials vaccination andchallengerdquo Refuah Veterinarith vol 39 pp 77ndash88 1982
ISRN Veterinary Science 5
[30] A J DaMassa D L Brooks C A Holmberg and A I MoeldquoCaprine mycoplasmosis an outbreak of mastitis and arthritisrequiring the destruction of 700 goatsrdquo Veterinary Record vol120 no 17 pp 409ndash413 1987
[31] P Kumar A Roy B B Bhanderi and B C Pal ldquoIsolationlsquoiden-tification and molecular characterization of Mycoplasma iso-lates from goats of Gujarat State Indiardquo Veterinarski Arhiv vol81 no 4 pp 443ndash458 2011
[30] A J DaMassa D L Brooks C A Holmberg and A I MoeldquoCaprine mycoplasmosis an outbreak of mastitis and arthritisrequiring the destruction of 700 goatsrdquo Veterinary Record vol120 no 17 pp 409ndash413 1987
[31] P Kumar A Roy B B Bhanderi and B C Pal ldquoIsolationlsquoiden-tification and molecular characterization of Mycoplasma iso-lates from goats of Gujarat State Indiardquo Veterinarski Arhiv vol81 no 4 pp 443ndash458 2011
