An Experimental Comparison of Methods for Somatic Cell Count Determination in Milk of Various Species of Mammals

HANUŠ, O., SOJKOVÁ, K., HANUŠOVÁ, K., SAMKOVÁ, E., HRONEK, M., HYŠPLER, R., KOPECKÝ, J., JEDELSKÁ, R.: An experimental comparison of methods for somatic cell count determination in milk of various species of mammals. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 1, pp. 67–82 Somatic cell count (SCC) is important foodstuff , hygienic and health indicator of milk and animal mammary gland. The goal of this paper was to evaluate an ability of chosen methods to reach the SCC reliable results in various biological kinds (species) of milk. The various methods of SCC determination were compared in cow (CM), goat (GM), sheep (SM) and human (HM) milk: direct microscopy (DM); fl uoro-opto-electronic (Fossomatic 90; Foss); fl uorescent (DCC; De Laval). Used methods had cow milk calibration basically. The DM, Foss and DCC result relations about SCC were very close, mostly > 0.92 (P < 0.001) for CM, GM and SM. In CM the regression equations between methods were near ideal form y = 1x + 0. The mean diff erences SCC data sets between mentioned methods were small for CM, larger for SM and HM and the largest for GM. It is possible to convert all DCC results in SM, HM and GM to DM or Foss method. The conversion equations were stated from DCC: to DM in cow milk y = 1.1293x − 5.5029; to Foss in goat milk y = 3.603x − 3171.4; to Foss in sheep milk y = 1.3805x − 18.149; to Foss in human milk y = 2.6246x + 158.63. Assesment of conversion equations should be individual laboratory event. Results had relatively good correspondence among DM, Foss and DCC for SCC determination in CM, GM, SM and HM for milk quality control. DCC had lower results in small ruminants as compared to Foss calibrated on CM using DM. DCC in HM had lower results as Foss adjusted by CM at good correlation (0.84; P < 0.001). cow, sheep, goat, human milk, somatic cell count (SCC), direct microscopy, fl uoro-opto-electronic method, fl uorescence method, relationship Somatic cell count (SCC) in milk is important food-hygienic (bulk samples) and also health (individual samples) indicator of animal mammary gland. Somatic cells are mostly leucocytes in milk (nucleus cells) which show on actual state of physiological and pathological response of animal defense system with respect to possible mammary gland infection. The SCC values show on milk secretion disor ders occurrence. Therefore the SCC determination serves over whole world to control of milk food chain quality in dairying and human nutrition from beginning of this succession. Critical quality values of SCC are limited by legislation for milk of some animal species. For cover of above mentioned facts more analytical methods are existing which are based on various principles, standardized and also unstandardized. Beside reference direct optical microscopy method (CSN EN ISO 13366–1 (DM)) there are also instrumental methods. Electronical counting of fi rm particules (somatic cells fi xed by formaldehyde) during their passage throught defi ned electrical fi eld in capillary was previous procedure (so called Coulter Counter method). Today other procedures such as fl uoro-opto-electronic counting (CSN EN ISO 68 O. Hanuš, K. Sojková, K. Hanušová, E. Samková, M. Hronek, R. Hyšpler, J. Kopecký, R. Jedelská 13366–2) of stained (ethidiumbromid) somatic cell nuclei (DNA) which emit red emission a er lighting which is registered electronically by disc rotation (DR, disc rotation; Fossomatic) method or more modern fl ow cytometry (FC, fl ow cytometry; for instance Fossomatic – Foss Electric, Denmark (Foss), Somacount – Bentley Instruments, USA or Somascope – Delta Instruments, The Netherlands) are used preferably. Also various modifi cations of viscosimetric methods (Wisconsin Mastitis test or Ruakura (New Zealand) rolling ball viscosimeter (Hanuš et al., 1993d)) on the principle of so called Schalm reaction (reaction among DNA, milk proteins and added detergent with creation of gel and its density increase at SCC rise; Hejlíček et al., 1987) are disposable for estimation of cell quantity as well. There are above all Schalm California mastitis test in modifi cation known as Mastitis tets NK in the Czech Republic. Recently a further instrumentation (Chemometec (2004), NucleoCounter SCC–100 and De Laval (2006), DCC cytometer, fl uorescent method principle (DCC)) is appearing, especially for direct advisory service purposes at fast and operative moni toring of udder health in animal stables (Castro et al., 2008; Sánchez–Macías et al., 2008). All these indirect instrumental methods of SCC determination should be calibrated or adjusted in other way according to direct microscop SCC determination results. Diff erences in result reliability regarding biological kind of milk can exist here. The row of papers was interested in methodical support of reference standard preparation for somatic cell count (SCC) determination in milk, especially in cows (Szijarto and Barnum, 1984; Lintner et al., 1984; Arndt et al., 1991; Heeschen et al., 1994; Aebi and Bühlmann, 2000a, b; Baumgartner et al., 2000). Also whole row of papers was interested in statistical evaluation of milk analytical result reliability in laboratory networks on national and international level (Vines et al., 1986; Grappin, 1987, 1993; Valenberg, 1990; Arndt et al., 1991; Leray, 1993, 2006, 2010; Wood, 1994; Heeschen et al., 1994; Golc–Teger, 1997; Wood et al., 1998; Baumgartner et al., 2000; Fuchs, 2000; Coveney, 2001; Feinberg and Laurentie 2006; Hanuš et al., 2006, 2007; Hering et al., 2008; Říha et al., 2008 – profi ciency testing (PT)). SCC determination in other biological kinds of milk in other farm animals (sheep, goats, buff alos, camels, horses or asses), mammals respectively, than in cows has growing importance. The main reason is an increase of herds of various animal species (Nicolas et al., 2008; Pellegrino and Rosi, 2008; Marchand et al., 2008) and also spectrum of milk food (for instance functional food) and its market. It means spreading of milk market and growth of an importance of food safety and consumer health control. The aim of this work was to examine pertinence of chosen analytical procedures to product reliable SCC results in various kind of milk which can methodically extend possibilities of control of hygiene standards and quality in dairying but especially in an animal health state. MATERIAL AND METHODS Model sample files of various biological kindsof milk Human milk samples were analysed as unpre-served. Other mammals species milk samples (ru-minants) were preserved (0.02%; Pettipher and Ro-drigues, 1982; Ardö, 1982; Kroger, 1985; Hanuš etal., 1992a, b; Genčurová et al., 1993a, 1994; Benda,1995) by peletted bronopol (2–brom, 2–nitro, 1, 3propandiol; Control System D&F; Microtabs, Eng-land). First and also second samples were analysedeither a er cold transport and storage (< 6 oC; ac-cording to Szijarto et al., 1990 and Sojková et al., 2009)or frozen preservation (−21 oC), according to condi-tions, however always in accordance with standardprocedure. Previous papers (Hanuš et al., 2002, 2008;Baumgartner and Landgraf, 2004) demonstratedalso possibility of milk sample frozen preserva-tion as usable procedure before analyse for obtain-ing reliable SCC results. Experimental comparisoncovered 1.5 lactation period of small ruminants byrealization time and in this way it took 1.5 year.I) cow milk (CM): reference (for PT), modified,mixed bulk and individual milk samples (n = 20,in two tests A and B) species Bos primigenius f. tau-rus (L), breeds Czech Fleckvieh and Holstein. Na-tive and frozen preserved samples, native sampleswere chemical preserved. The used methods of SCCdetermination were DM (proficiency testing, Fig. 1with good result), Foss and DCC.II) goat milk (GM): bulk milk samples (n = 30samples, 3 tests A, B and C) from small count of ani-mals (from 4 to 6 animals in one sample) speciesCapra aegagrus f. hircus (L), breed White short haired,from morning milking. Native milk samples weresubsequently chemical preserved and also frozenpreserved. Used methods of SCC determinationwere Foss and DCC.III) sheep milk (SM): bulk milk samples (n = 40samples, 4 tests A, B, C and D) from small count ofanimals (from 4 to 6 animals in one sample), speciesOvis aries (L), greed Tsigai, from morning milking.Native milk samples were subsequently chemicalpreserved and also frozen preserved. Used methodsof SCC determination were Foss and DCC.IV) human milk (HM): individual milk samples(n = 54 samples, 1 test A), species Homo sapiens sapiens(L), originated from primipar mothers in age from23 to 27 years. Samples were taken during 1.5 year,from 2nd to 47th lactation week, always in morninghours a er 12 hunger hours. Whole volume of milkwas exhausted from breast which was not suckledlast time. The sample obtained in this way was fro-zen preserved before analyse. Used methods of SCCdetermination were Foss and DCC. Examined analytical methods for somatic cellcount determination in milk The direct microscopy method (DM) was usedfor counting of stained somatic cells (CSN EN ISO An experimental comparison of methods for somatic cell count determination in milk69 13366–1) at SCC determination in CM referencestandards for proficiency testing (PT) purposes.Milk samples from all species (CM, GM, SM,HM) were analysed on SCC using fl uoro–opto–electronic method in DR type (Foss) on Fosso-matic 90 (Foss Electric, Denmark) apparatus withregard to previous methodical knowledge (Grap-pin and Jeunet, 1974, 1975; Coleman and Moos,1989; Genčurová et al., 1993b; Hanuš et al., 1993a,b, c, 2002, 2007, 2009) and according to CSN ENISO 13366–2. This apparatus was included in pro-ficiency testing (State Veterinary Institute, Prague)for SCC determination regularly three times a year.The results were regularly in order (Fig. 1). The ex-tended combine result uncertainty (Suchánek etal., 1999) of measurement was estimated in accred-ited laboratory about ± 9.3% for SCC < 900103.ml−1.The mentioned SCC result assurance was in accor-dance with approaches which were published byValenberg (1990), Arndt et al. (1991), Grappin (1993),Leray (1993), Heeschen et al. (1994), Wood (1994),Golc–Teger (1997), Wood et al. (1998), Baumgartner(2000), Fuchs (2000), Aebi and Bühlmann (2000a,b), Coveney (2001), Feinberg and Laurentie (2006)and Hanuš et al. (2007). Further, there is necessary tobring out that all used indirect SCC determinationmethods in this paper were primarily adjusted forcow milk analyses.Also samples of all milk kinds were analysed onSCC using fluorescent method (DCC; fluorescentsignal measurement a er DNA staining by propid-ium iodine) according to producer instruction man-ual (De Laval, 2006; Sweden). The relevant method-ical information about method principle capabilityare included in other professional papers (Chemom-etec, 2004; Denmark). Relationships of instrumen-tal SCC determination in milk to direct microscopicmethod or reference values from profi ciency testing(for instance CECALAIT) were demonstrated. Thelinear regression relationship had form for exampley = 1.05 + 15.78 for CM.The analyses were performed in accredited Na-tional Reference Laboratory for Raw Milk (accord-ing to CSN EN ISO/IEC 17025) which is situated inResearch Institute for Cattle Breeding in Rapotín(n. 1340, certificate n. 040/2005) and which co-op-erates in network of national reference laboratoriesfor milk under AFSSA (Agence Francaise de Sécu-rité Sanitaire des Aliments) Paris supervision. Inthe case of high SCC in HM (> 300103.ml−1) a bac-teriological examination on occurrence of mastitispathogens was performed simultaneously (proce-dures according to: Hejlíček et al., 1987; Benda andVyletělová, 1995, 1997a, b; Benda et al., 1997). Statistical evaluation The relationships between SCC determination re-sults by various methods were evaluated using ba-sic methods of diff erence statistics (set mean (x) andits standard deviation (sd), mean difference (d) andits variability (standard deviation, dsd) and usinglinear and nonlinear regression (Grappin and Jeu-1: Regular proficiency test graph for somatic cell counters (fluoro–opto–electronic technology by disc rotation (DR) or flow cytometry (FC)) incow milk in the Czech Republic (n = 16)(SVI Prague, NRL-M, authorized so ware SomaRing, AgriResearch Rapotin, Říha et al., 2008; used apparatus Fossomatic90 in National Reference Laboratory for Raw Milk in methodical comparison (offi cial proficiency testing) from period ofmethod comparison of SCC determination on various biological kinds of milk is marked) 70O. Hanuš, K. Sojková, K. Hanušová, E. Samková, M. Hronek, R. Hyšpler, J. Kopecký, R. Jedelská net, 1974, 1975; Grappin, 1987; Hanuš et al., 1993a,b, c, 2002, 2006, 2007, 2009). Linear form is hereatpreferred as it is known by generally accepted cali-bration model for indirect methods in link to directmethods of milk analyses (Grappin, 1987; Baumgart-ner, 2006; Leray, 2006, 2010; Hanuš et al., 2009). SCCswere evaluated in original values and also in loga-rithmic (log10) transformed form (log SCC) becauseof presupposed occurrence of lognormal frequencydistribution in individual milk samples (Ali andShook, 1980; Shook, 1982; Raubertas and Shook,1982; Reneau et al., 1983 and 1988; Reneau, 1986;Wiggans and Shook, 1987; Hanuš et al., 1995; Janůet al., 2007). Therefore also geometrical mean values(xg; mean logarithm a er reverse transformation in103.ml−1) could be used for comparison between in-dividual methodical SCC files. Also pair t-test wasused for testing of diff erences between SCC meansthough the interpretation of these results has to becareful from analytical point of view because it canbe misguided. Nevertheless, in the same time undunder mentioned presupposition the pair t-testis used in result graphical interpretation of profi -ciency testing with indicator of Euclidian distancefrom origin (Leray, 1993, 2006 and 2010; Hanuš et al.,1998, 2006 and 2007). In case that result repeatabil-ity of SCC measurement by various methods werecalculated in data fi les as standard deviations it wascarried out according to papers Grappin (1987) andHanuš et al. (1998) by repeated SCC measurementin various milk samples. The Microso Excel pro-gramme was used for calculations. RESULTS AND DISCUSSION