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h p://doi.o g/10.1016/j.idai yj.2014.09.007
h p://hdl.handle.ne /10251/80581
Else ie
Bel án Ma ínez, MC.; Be uga Fe nandez, MI.; Molina Casano a, A.; Lisand o Al haus, R.;
Molina Pons, MP. (2015). Pe o mance o cu en mic obial es s o sc eening an ibio ics in
sheep and goa milk. In e na ional Dai y Jou nal. 41:13-15. doi:10.1016/j.idai yj.2014.09.007.
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Pe o mance o cu en mic obial es s o sc eening an ibio ics in sheep and
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goa milk
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Bel án M.Ca., Be uga M.Ib., Molina Ab., Al haus R.Lc., Molina M.Pa.
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aIns i u o de Ciencia y Tecnología Animal, Uni e si a Poli ècnica de València, Camino de
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Ve a, s/n, 46022 Valencia, Spain. [email p o ec ed]; [email p o ec ed]
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bDepa amen o de Ciencia y Tecnología Ag o o es al, ETSIA-IDR, Uni e sidad de Cas illa-
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La Mancha, 02071 Albace e, Spain. [email p o ec ed]; [email p o ec ed]
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cCá ed a de Bio ísica, Facul ad de Ciencias Ve e ina ias, Uni e sidad Nacional del Li o al,
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R.P.L., K ede , 3080 Espe anza, A gen ina. [email p o ec ed]
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Co esponding au ho : Ma ia Ca men Bel án Ma ínez
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Ins i u o de Ciencia y Tecnología Animal
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Uni e si a Poli ècnica de València
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Camino de Ve a, s/n
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46022 Valencia, Spain
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Tel.: + 34 963877727; Fax: +34 963877439
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[email p o ec ed]
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ABSTRACT
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The de ec ion capabili y (CCβ) o some mic obial sc eening es s (BRT MRL, Del o es
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MCS SP-NT, Del o es MCS Accele a o and Eclipse 100) cu en ly a ailable was
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calcula ed in acco dance wi h Commission Decision 657/2002/EC. The CCβ was a o
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below he maximum esidue limi (MRL) o mos β-lac ams assessed and o he non-β-
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lac am d ugs such as neomycin, ylosin, sul adiazine and sul adime hoxine. Howe e ,
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he es s we e less sensi i e in he de ec ion o mos non-β-lac am d ugs such as
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quinolones and e acyclines a sa e y le els. When indi idual sheep milk samples ee
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o an ibio ics we e analysed, an ele a ed soma ic cell coun (SCC) was ela ed o he
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occu ence o non-complian esul s in all he me hods assessed. In o de o gua an ee
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he sa e y o milk and dai y p oduc s om small uminan s, he pe iodical
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implemen a ion o sc eening es s mo e sensi i e owa ds non-β-lac am d ugs would be
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con enien .
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1. In oduc ion
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Mic obial inhibi o es s a e ou inely applied o sc eening an ibio ics in aw milk as
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hey a e ela i ely inexpensi e, use - iendly, and ha e a high sample h oughpu .
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Mos cu en mic obial sc eening es s we e ini ially de eloped o de ec β-lac ams in
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cow milk and a e based on he inhibi ion o Geobacillus s ea o e mophilus a .
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calidolac is being highly sensi i e o hese subs ances. Se e al s udies on he mic obial
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es sensi i i y using sheep milk ha e been ca ied ou in he las wo decades (Al haus,
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To es, Mon e o, Balasch & Molina, 2003a; Molina, Al haus, Molina & Fe nández,
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2003), while e y ew s udies ha e been unde aken in goa milk (Sie a e al., 2009a,b),
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demons a ing ha hese es s a e able o de ec β-lac ams a o below he Maximum
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Residue Limi s (MRLs) es ablished by Eu opean legisla ion (EC, 2010), bu canno
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sui ably de ec o he e e ina y d ugs.
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The e o e, modi ica ions such as he addi ion o chela ing agen s in o he cul u e
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medium o enhance he de ec ion o e acyclines, espec i ely, o an i ola es o imp o e
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sulphonamide de ec ion ha e been p oposed (Lange eld, Beuke s, Bommele, & S a k,
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2005), and consequen ly manu ac u e s ha e imp o ed some pe o mance
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cha ac e is ics o mic obial sc eening es s in new e sions now a ailable.
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Mo eo e , sheep and goa milk is cha ac e ised by a highe a and p o ein con en han
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cow milk (Pa k, Jua ez, Ramos & Haenlein, 2007), an ele a ed na u al inhibi o con en
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(e.g. immunoglobulins, lac o e in, o lysozyme) (C osson, Thomas, & Rossi, 2010) and
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a highe soma ic cell coun (SCC) e en in he absence o in a-mamma y in ec ions
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(Medhid, Díaz, Ma í, Vidal & Pe is, 2013), po en ially in e e ing in he mic obial
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inhibi o es esponse.
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Sheep and goa milk p oduc ion is mainly des ined o he elabo a ion o dai y p oduc s.
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An ibio ic esidues in milk may pa ially o o ally inhibi e men a ion p ocesses in
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cheese and yogu p oduc ion (Packham, B oome, Limsow in, & Roginski, 2001).
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Mo eo e , consume sa e y may be comp omised by he p esence o hese esidues in
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dai y p oduc s (Oli e , Mu inda & Ja ayao, 2011). Thus, he aim o his s udy was o
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assess he pe o mance o new e sions o some mic obial sc eening es s o de ec
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an imic obial esidues in sheep and goa milk acco ding o Eu opean Commission
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Decision nº 657/2002 (EC, 2002).
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2. Ma e ial and me hods
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2.1. Mic obial inhibi o es s: The sc eening es s used we e he BRT MRL (Analy ik in
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Milch P oduk ions-und Ve iebs-GmbH. Munich, Ge many), Del o es MCS SP-NT
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(DSM Food Special ies. Del , he Ne he lands), Del o es MCS Accele a o (DSM
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Food Special ies), and Eclipse 100 (Zeu-Inmuno ec. Za agoza, Spain). All es s we e
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conduc ed acco ding o each manu ac u e ’s ins uc ions.
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2.2. Milk samples: An ibio ic- ee milk samples we e ob ained om he expe imen al
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locks o Manchega sheep o Uni e sidad de Cas illa-La Mancha (Albace e, Spain), and
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Mu ciano-G anadina goa s o Uni e si a Poli ècnica de València (Valencia, Spain).
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Animals had a good heal h s a us and had no ecei ed any e e ina y d ugs, nei he
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be o e no du ing he expe imen al pe iod, no was medica ed eed used in hei die . All
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milk samples we e analysed o g oss composi ion (MilkoScan 6000, Foss. Hille d,
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Denma k), soma ic cell coun (Fossoma ic 5000, Foss), o al bac e ial coun (Bac oscan
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FC, Foss), and pH alue (pHme e , C ison, Ba celona, Spain).
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2.3. An imic obials and spiked milk samples: A o al o 37 subs ances was in es iga ed:
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amoxicillin, ampicillin, benzylpenicillin, cloxacillin, dicloxacillin, na cillin, oxacillin,
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ce alonium, ce api in, ce azolin, ce ope azone, ce quinome, ce io u , cephalexin,
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chlo e acycline, cip o loxacin, colis in, en o loxacin, e y h omycin, gen amicin,
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lincomicin, ma bo loxacin, neomycin, oxy e acycline, s ep omycin, sul adiazine,
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sul adime hoxine, sul ame azine, e acycline, ime hop im, and ylosin we e p o ided
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by Sigma-Ald ich Química, S.A. (Mad id, Spain). Des u oylce io u was supplied by
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To on o Resea ch Chemicals, Inc. (To on o, Canada) and he 4-epime s o e acyclines
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we e u nished by Ac os O ganics (Geel, Belgium). Finally, desace ylce api in and
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ce ace ile, no comme cially a ailable, we e kindly p o ided by Fa o S.p.A. (Bologna,
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I aly) and ACS Dob a , S.p.A. (Milan, I aly), espec i ely. Spiked milk samples we e
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p epa ed ollowing In e na ional Dai y Fede a ion ecommenda ions (ISO/IDF, 2003),
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and es ed by he mic obial sc eening es s immedia ely a e spiking.
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2.4. De ec ion capabili y (CCβ): Tes de ec ion capabili y (CCβ) was in es iga ed
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ollowing he “Guidelines o he alida ion o sc eening me hods o esidues o
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e e ina y medicines” p oposed by Communi y Re e ence Labo a o ies o esidues
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(CRLs, 2010), supplemen ing Commission Decision nº 657/2002 (EC, 2002).
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An imic obial- ee milk samples we e spiked indi idually wi h di e en subs ances a
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0.5·MRL, 0.75·MRL, and 1·MRL equi alen d ug concen a ion, and analysed 20, 40 o
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60 imes, espec i ely, by he di e en mic obial es s.
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2.5. In e e ences ela ed o milk ma ix cons i uen s: An ibio ic- ee milk samples we e
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ob ained on a wo-week basis along he en i e milking pe iod (sheep: n=250, sampling
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days 30 o 180 pos -pa um; goa s: n=350, sampling days 15 o 200 pos -pa um) and
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analysed simul aneously, in h ee eplica es, by he ou es s. The e ec o he milk
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ma ix cons i uen s on he es esponse was in es iga ed using he s epwise op ion o
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he logis ic p ocedu e o he SAS so wa e, acco ding o he ollowing model:
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Lij= logi [Pi] = β0 + β1[SL] + β2[pH] + β3[F] + β4[P] + β5[L] + β6[TS] + β7[logSCC] +
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β8[logBC] + εij (Eq. 1)
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whe e: Lij= logis ic model; [Pi]= p obabili y o he esponse ca ego y (posi i e/nega i e);
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β0= in e cep ; βi= es ima e pa ame e s o he model; [SL]= lac a ion s age e ec (day);
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[pH]= pH e ec ; [F]= a con en e ec ; [P]= p o ein con en e ec ; [L]= lac ose con en
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e ec ; [TS]= o al solids con en e ec ; [logSCC]= soma ic cell coun e ec ; [logBC]=
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bac e ial coun e ec ; εij= esidual e o .
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3. Resul s and discussion
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3.1. De ec ion capabili y (CCβ): The CCβ o he BRT MRL, Del o es SP-NT,
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Del o es DA, and Eclipse 100 es s in sheep and goa milk we e shown in Table 1. In
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gene al, mic obiological es s de ec ed high equencies o β-lac am an ibio ics in milk
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om sheep (70.6 %: BRT MRL, and 88.2 %: Del o es SP-NT, Del o es DA, and
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Eclipse 100) and goa s (76.4 %: BRT MRL, and 82.3 %: Del o es SP-NT; Del o es
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DA, and Eclipse 100). Only ce quinome and ce ope azone could no be de ec ed by any
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es a MRL equi alen an ibio ic concen a ion.
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Fo non-β-lac am d ugs, he mic obiological es s could no de ec ed esidues o
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quinolones, e acyclines, s ep omycin, lincomicin, sul ame azine, colis in and
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ime hop im a sa e y le els. Con e sely, neomycin, ylosin sul adiazine and
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sul adime oxine we e de ec ed a o below egula o y limi s (Table 1). Mo eo e , he
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BRT MRL es was also able o de ec gen amicin and e y h omycin a hei MRLs.
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3.2. In e e ences ela ed o he milk ma ix e ec
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Indi idual milk, ee o an imic obials, p esen ed a wide ange o a ia ion o all milk
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quali y pa ame e s conside ed. When indi idual sheep milk samples we e analysed an
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ele a e pe cen age o non-complian esul s we e ob ained (BRT MRL: 4.8 %,
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Del o es SP-NT: 8.0 %, Del o es DA: 10 % and Eclipse 100: 9.6 %). Applying
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logis ic eg ession analysis, an inc ease in SCC was associa ed wi h an ele a ion in he
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p edic ed likelihood o posi i e ou comes in all cases (Figu e 1), he BRT MRL es
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esponse being he less a ec ed by his pa ame e . These esul s we e in ag eemen wi h
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hose ob ained by Cullo e al. (1992) and Al haus e al. (2003b) using indi idual milk
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samples om cows and sheep, espec i ely. Ins ead, he pe cen age o non-complian
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esul s in goa milk we e lowe in all cases (BRT MRL: 1.4 %, Del o es SP-NT: 4.3 %,
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Del o es DA: 3.1 % and Eclipse 100: 0.6 %), and he e o e, logis ic p ocedu e was no
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pe o med.
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4. Conclusions
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Mic obial inhibi o es s a e e icien o de ec β-lac ams and o he non-be a-lac am
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d ugs such as neomycin, ylosin, sul adiazine and sul adime hoxine in aw milk om
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small uminan s. Howe e , in spi e o he imp o emen s made in hese es s in he las
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decade, hey con inue o be ine icien o he de ec ion o o he d ugs, such as
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quinolones and e acyclines, a sa e y le els. The e o e, he pe iodic use o mo e
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sensi i e es s owa ds hese subs ances would be con enien o widen he de ec ion
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ange in sc eening and gua an ee he quali y o milk and dai y p oduc s om small
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uminan s.
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5. Acknowledgemen s
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This esea ch o ms pa o he P ojec AGL2009-11524 inanced by he Minis e io de
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Ciencia e Inno ación (Mad id, Spain).
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6. Re e ences
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Al haus, R.L., To es, A., Mon e o, A., Balasch, S., & Molina, M.P. (2003a). De ec ion
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limi s o an imic obials in ewe milk by Del o es pho ome ic measu emen s.
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Jou nal o Dai y Science, 86, 457-463.
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Al haus, R.L., To es, A., Pe is, C., Bel án, M.C., Fe nández, N, & Molina M.P.
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(2003b). Accu acy o BRT and Del o es mic obial inhibi ion es s as a ec ed by
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composi ion o ewe’s milk. Jou nal o Food P o ec ion, 66, 473-478.
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CRLs. (2010). Guidelines o he alida ion o sc eening me hods o esidues o
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e e ina y medicines. Communi y Re e ence Labo a o ies o esidues. A ailable a
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h p://ec.eu opa.eu/ ood/ ood/chemicalsa e y/ esidues/lab_analysis_en.h m (Las
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upda e: 20-01-2010).
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C osson C., Thomas, D., & Rossi, C. (2010). Quan i ica ion o Immunoglobulin G in
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bo ine and cap ine milk using a su ace plasmon esonance-based immunosenso .
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Jou nal o Ag icul u al and Food Chemis y, 58, 3259-3264.
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Cullo , J.S., Van Eenennaam, A., Dellinge , J., Pe ani, L., Smi h, W., Jensen, L. (1992).
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An ibio ic esidue assays: can hey be used o es milk om indi idual cows?.
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Ve e ina y Medicine, 87, 477-494.
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EC. (2002). Commission Decision 2002/657/EC o 12 Augus 2002 implemen ing
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Council Di ec i e 96/23/EC conce ning he pe o mance o analy ical me hods and
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he in e p e a ion o esul s. O icial Jou nal o Eu opean Communi ies, L 221, 8-36.
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EC. (2010). Regula ion (EU) Nº 37/2010 o 22 Decembe 2009 on pha macologically
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ac i e subs ances and hei classi ica ion ega ding maximum esidue limi s in
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oods u s o animal o igin. O icial Jou nal o Eu opean Communi ies, L 15, 1-72.
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ISO/IDF (2003). Milk and milk p oduc s- Guidelines o a s anda dized desc ip ion o
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mic obial inhibi o es s. IDF S anda d Nº183:2003, B ussels, Belgium:
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In e na ional Dai y Fede a ion.
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Lange eld, P.C., Beuke s, R., Bommele, M.W.C., & S a k, J. (2005). Rapid
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mic obiological es o he de ec ion o an ibac e ial compounds. Pa en Nº US
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6,867,015B. US Pa en Ma . 15, 2005.
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Medhid A., Díaz J.R., Ma í A., Vidal G., Pe is C. 2013. E ec o es us
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synch oniza ion on daily soma ic cell coun a ia ion in goa s acco ding o lac a ion
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numbe and udde heal h s a us. Jou nal o Dai y Science, 96:4368–4374.
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Molina, M.P., Al haus, R.L., Molina, A., & Fe nández, N. (2003). An imic obial agen
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de ec ion in ewe’s milk by he mic obial inhibi o es b illian black educ ion es -
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BRT AiM®. In e na ional Dai y Jou nal, 13, 821-826.
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Oli e , S.P., Mu inda, S. E., & Ja ayao, B.M. 2011. Impac o an ibio ic use in adul
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dai y cows on an imic obial esis ance o e e ina y and human pa hogens: a
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comp ehensi e e iew. Foodbo ne Pa hogens and Disease, 3, 337-355.
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Packham, W., B oome, M.H., Limsow in, G.K., & Roginski, H. (2001). Limi a ions o
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s anda d an ibio ic sc eening assays when applied o milk o cheesemaking. Aus alian
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Jou nal Dai y Technology, 56, 15–18.
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Pa k, Y.W., Jua ez, M., Ramos, M., & Haenlein, G.F.W. (2007). Physico-chemical
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cha ac e is ics o goa and sheep milk. Small Ruminan Resea ch, 68, 88-113.
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Sie a, D., Sánchez, A., Con e as, A., Luengo, C., Co ales, J.C., Mo ales, C.T., De la
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Fe, C., Gui ao, I., & Gonzalo, C. (2009a). De ec ion limi s o ou an imic obial
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