J. Dairy Sci. 84(E. Suppl.):E237-E244 The American Dairy Science Association, 2001.

Milk Somatic Cells and Lactation in Small Ruminants

M. J. Paape,*Bernard Poutrel, Antonio Contreras, Juan C. Marco, and A. V. Capuco
*

Immunology and Disease Resistance Laboratory, USDA-ARS, Beltsville, MD, 20705, Insitut National de la Recherche Agronomique, Centre de Recherches de Tours, 37380 Nouzilly, France Enfermedades Infecciosas, Facultad de Veterinaria, Universidad de Murcia, 30071, Murcia, Spain, Laboratorio Normativo de Salud Pdblica de Bilbao, Maria Diaz de Haro, 60, 48010-Bilbao, Spain, Gene Evaluation and Mapping Laboratory, USDA-ARS, Beltsville, MD, 20705

ABSTRACT

The milk somatic cell count (MSCC) is the basis for abnormal milk control programs. The current legal MSCC limit for bulk tank milk for goats and sheep in the United States is 1000 and 750 103/ml, respectively. Milk somatic cell counts for goats are higher than MSCC for cows and sheep. The MSCC for goats free from intramammary infection (IMI) range from 270 to 2,000 103/ml. Cell counts for sheep are similar to cows and range from 10 to 200 103/ml. Neutrophils comprise the major cell type in milk from uninfected goats and constitute 45 to 74% of the MSCC, compared with 2 to 28% for sheep and cows. The macrophage is the major cell type in milk from cows and sheep. Milk secretion in goats and sheep is largely apocrine in nature and cytoplasmic particles, similar in size to milk somatic cells, are normal constituents of their milk. Concentrations of cytoplasmic particles in sheep milk average 15 103/ml, while goat milk averages 150 103/ml. Therefore, to obtain accurate MSCC for goats, only cell counting procedures specific for DNA should be used. While IMI significantly increases MSCC for goats and sheep, noninfectious factors such as parity, stage of lactation, season and milk yield have been related to increased MSCC. An increase in MSCC for goats has been shown to decrease milk and fat yields. Intramammary infusion of antibiotics at dry-off and postmilking teat dipping in goats decreased the rate of new IMI and MSCC. Thus, mastitis control practices shown to be efficacious in cows are also effective in goats. (Key words: milk somatic cell count, goat, sheep, mastitis). Abbreviation key: CAEV = caprine arthritis encephalitis virus, CMT = California mastitis test, CNS = coagulasenegative staphylococci, DMSCC = direct microscopic SCC, EU = European Union, M/V = Maedi/Visna, MSCC = milk SCC, PMN = polymorphonuclear neutrophilic leukocyte. Total and Differential Milk SCC Goats. The milk SCC (MSCC) for uninfected goats are higher than MSCC for uninfected cows and sheep (Paape and Capuco, 1997). On average, MSCC for goat mammary glands

free from IMI range from 270 to 2000 103/ml and 659 to 4213 103/ml for infected glands (Table 1). Unlike milk from cows and sheep, polymorphonuclear neutrophilic leukocytes (PMN) comprise the major cell type in milk from infected and uninfected goat mammary glands (Dulin et al., 1983). For animals free of IMI, PMN constitute 45 to 74% of the somatic cells in goat milk and 71 to 86% for infected mammary glands. Macrophages comprise 15 to 41% of the somatic cells in uninfected halves and 8 to 18% in infected halves. Lymphocytes comprise 9 to 20% of the somatic cells in uninfected halves and 5 to 11% in infected halves. Epithelial cells represent a small portion of cells in goat milk; but identification by light microscopy is difficult because of the presence of cytoplasmic particles in goat milk. An early study (Dulin et al., 1982) reported that epithelial cells comprised less than 1% of the total cells. More recent studies reported that 6% of the cells in uninfected mammary glands were epithelial in origin (Contreras et al., 1998). Because milk secretion in the goat is
Table 1. Results of milk SCC scores for healthy and infected goat udder halves.1,2 Cells 103/ml Reference Dulin et al., 1982 Dulin et al., 1983 Poutrel and Lerondelle, 1983 Timms and Schultz, 1985 Lerondelle et al., 1992 Kalogridou-Vassiliadou et al., 1992 Wilson et al., 1995 Contreras et al., 1996 Type AM GM AM GM AM AM Uninfected 0280 0481 0614 0337 0520 0270 Infected 1690 1778 1293 0659 1040CNS 3800

Received August 1, 2000. Accepted October 1, 2001. Corresponding author M. J. Paape; e-mail: maape@lpsi.barc.usda. gov.

LS 0303650 373800 GM 0396 0873 AM 1115 1909 Corrales et al., 1996 AM 1235 2186 De Cremoux et al., 1996 GM 0493 1078CNS2731MP AM 0973 1564CNS3591MP Ferrer et al., 1996 AM 0349571 19003393 Le Mens et al., 1996 GM 1059 2511 Poutrel et al., 1996 GM 0272 0932CNS2443MP AM 0687 1462CNSr4213MP Snchez et al., 1996 GM 0341 1218 AM 0938 2147 Vihan, 1996 AM 0332 0708 Contreras et al., 1997 GM 2000 2500 1 Adapted from Snchez et al. (1998). 2 AM = Arithmetic mean; GM = geometric mean; LS = linear score; CNS = udder halves infected with coagulase-negative staphylococci; MP = udder halves infected with major pathogens. Vol. 84, E. Suppl., 2001 E237

SYMPOSIUM: SMALL RUMINANT DAIRY RESEARCH AND PRODUCTION

Table 2. Comparison of methods for estimating SCC in goat milk.1 Method No. of cells ( 105/ml)2 Pyronin Y-methyl green stain 3.40a Fossomatic cell counter 3.65ab Wisconsin mastitis test 4.94bc Coulter Counter 6.44cd Levowitz-Weber stain 7.92d Significance level P < 0.01 Standard error 1.1324 1 Adapted from Dulin et al. (1982). 2 Each value represents the mean of 24 determinations run in duplicate. Means with the same superscript letter in common are not significantly different (P < 0.05).

Figure 1. Transmission electron micrograph section through a biopsied sample from the mammary gland. The alveolar lumen is bound by epithelial cells whose apex has external microvilli and internal granular accumulation in the endoplasmic reticulum that resemble those found in the cytoplasmic particles of milk. Bar = 10 Pm.

mainly apocrine (Wooding et al., 1970), cytoplasmic particles are shed into milk from the apical portion of mammary secretory cells (Figure 1). The number of cytoplasmic particles in milk of uninfected mammary halves range from 71 to 306 103/ml and 98 to 231 103/ml for infected mammary glands (Dulin et al., 1983). Although the majority of these particles are generally anucleated, approximately 1% have been observed to contain nuclear fragments (Dulin et al., 1982). Sheep. The MSCC and types of cells in milk from ewes free of IMI are very similar to those observed for cows (De la Cruz et al., 1994; Gonzalez-Rodriguez et al., 1995; Romeo et al., 1996). In a study by Romeo et al. (1996), MSCC for half udders of ewes free from IMI throughout lactation averaged 185 103 cells/ml of milk and 1445 103 cells/ml for infected halves. Halves with recurring infections averaged 576 103/ml. Regardless of infection status, counts increased with advancing lactation. The macrophage is the predominant cell type (46 to 84%) in milk from uninfected mammary glands. The PMN comprise 2 to 28% of the cell population and lymphocytes 11 to 20%. Plasma cells are present in small numbers in colostrum (0 to 20%), as are epithelial cells (1 to 2%). For infected mammary glands, the percentage of PMN increases to 50% at a MSCC of 200 103/ml and to 90% at a MSCC over 3 106/ml (Cuccuru et al., 1997). Cytoplasmatic particles are normal constituents in ewe milk and colostrum. However, concentrations are 10 times less than counts in goat milk, averaging 15 103cells/ml (Martinez et al., 1997). Enumeration of Somatic Cells in Milk Methodological requirements differ for MSCC determination of goat and sheep milk. Only cell counting procedures that are specific for DNA (fluoro-optical electronic cell counter, MSCC using DNA specific stains) should be used to estimate SCC in goat milk, because of the presence of larger anucleated cytoplasmic particles in goat milk (Dulin et al.,
E238 Journal of Dairy Science

1982; Table 2). Results from that study (Dulin et al., 1982) indicated no significant difference between MSCC stained with pyronin Y-methyl green and the Fossomomatic (fluoro-optical electronic counter) cell counts. The Wisconsin Mastitis Test, which is also DNA-specific, did not differ significantly from the Fossomatic cell counts. Coulter electronic cell counts and direct microscopic SCC (DMSCC) stained with LevowitzWeber gave significantly higher values. The California Mastitis Test (CMT), an indirect measure of somatic cells in milk can be used as a screening test for goat and ewe milk (Schalm et al., 1971; Schalm and Noorlander, 1957). The basis of the CMT reaction is DNA from somatic cells (Carroll and Schalm, 1962; Paape et al., 1962). The reagent used in the CMT consists of the detergent sodium alkylarylsulfonate, sodium hydroxide and a pH indicator. The detergent lysis the somatic cells in milk releasing DNA into the solution of sodium hydroxide, causing formation of a gel. Particle counters such as the Coulter electronic cell counter and DMSCC using stains that are not DNA-specific should not be used for goat milk (Dulin et al., 1982). Although milk secretion in ewes also consists of a large apocrine component, the concentration of cytoplasmic particles are one-tenth the counts in goat milk, averaging 15 103/ml (Martinez et al., 1997). Thus, electronic particle counters and stains not specific for DNA can be used for ewe milk. When using fluor-optical electronic cell counts, assays should be performed 1 to 5 d after collection on preserved milk samples maintained at 40C during counting. For freshly collected milk samples, heating for 15 min at 60oC is necessary to allow staining of the DNA with the fluorescent dye ethidium bromide (Miller et al., 1986). Milk preserved with azidiol, which is used in various European countries, will result in an underestimation of MSCC. In one study, MSCC for ewe milk preserved with either azidiol, bronopol, or potassium dichromate averaged (geometric mean) 501, 542, and 536 103/ml, respectively, (Gonzalo and San Primitivo, 1998). Goat milk standards are available for calibration of electronic cell counters (Dairy Quality Control Institute Services, Mountain View, MN). For sheep milk, the current standard used for calibration of electronic cell counters is cow milk with a MSCC range from 200 103 to 800 103/ml. However, this range does not adequately cover the wide range in MSCC in milk from ewes with subclinical IMI (Gonzalo and San Primitivo, 1998). A MSCC standard for the calibration of fluorooptical cell counters can be prepared as previously described for cow milk (Corlett et al., 2000). Acting in response to a need to develop a reliable procedure for the direct microscopic counting of cells in milk, the sub-

PAAPE ET AL.

committee on Screening Tests of the National Mastitis Council was formed (National Mastitis Council, 1996). The DMSCC was developed to meet the need for a precise standardized method to be used in abnormal milk control programs that were being implemented in the United States and European Union (EU) in the early 1970s (Lawton and Packard, 1972). The procedure for performing the DMSCC is as follows. Milk (0.01 ml) is spread onto a circular area (1 cm2) marked on the surface of a clean glass slide (Bellco Glass, Inc., Vineland, NJ; catalog #5638-01930) and allowed to dry on a level surface. For counting cells in ewe milk, the dried milk smears are stained for microscopic counting using any one of three Levowitz-Weber stains, a Canadian modification of the modified Newman-Lampert stain or the pyronin Y- methyl green stain (Packard et al., 1992). Because of the large number of cytoplasmic particles in goat milk, the pyronin Y-methyl green staining procedure is the official stain in the United States for performing DMSCC in goat milk (Packard et al., 1992). The ocular of the microscope should contain an ocular reticle (Dairy Research Production, Inc., Yarmouth, ME). The reticle has a wide and narrow strip centrally located and perpendicular to each other. The wide strip is used for low cell count milk and the narrow strip for high cell count milk. Thus, each microscope field is divided into four quadrants to facilitate enumeration of somatic cells. The microscope for the stripcounting procedure is calibrated as follows: 1) Measure the width of the wide and narrow strips in the reticle with a stage micrometer slide, ruled in 0.1- to 0.01-mm divisions. 2) A 10 ocular with a 1.8-mm oil-immersion objective is preferred. Calculate the strip area. The area of a single strip is equal to the width of the strip (in mm) times the length of the milk film, which is 11.28 mm. 3) Determine the number of single strips in the 0.01-ml milk film by dividing 100 mm2 (area of 0.01-ml milk film) by the strip area. The number of single strips = 100 mm2/area of a single strip. 4) Convert the number of single strips in 0.01 ml of milk to 1.0 ml by multiplying by 100. 5) Determine conversion factors for wide and narrow strips separately for each microscope using the following example: Length of strip (diameter of 1 cm2 circle) = 11.28 mm Width of wide strip in reticle = 0.089 mm (for example) Equation: Total area (100 mm2) dilution factor (100)/ (Area counted (11.28 mm 0.089 mm)/Number of strips counted) = 9961 Thus, each cell counted in the wide strip equals 9961 cells. If one counts 10 cells within the strip across the center of an entire milk smear, the cell count is 99,610 cells/ml milk. If 25 cells are counted within two wide strips across the horizontal and vertical central portions of the smear, the cell count is 25 cells 4980 or 124,500 cells/ml. A table, such as the one below, can be constructed to pro-

vide conversion factors for the number of cells counted within wide or narrow strips for the microscope in your laboratory. No. strips counted 01 02 03 04 01 02 03 04 Strip size Wide Wide Wide Wide Narrow Narrow Narrow Narrow Conversion factor 9961 4980 3320 2490 29,551 14,775 9850 7388

Milk smears to be stained with pyronin Y-methyl green need to be placed overnight in a desiccator. This is to assure that the milk smears are adhered firmly to the glass slide and will not wash off during staining. Smears are stained using a modification of the procedure as described by Paape et al., (1963). The smears are fixed for 10 min in Carnoys fixative (1 part glacial acetic acid: 3 parts chloroform: 6 parts 100% ethanol), then are hydrated for 2 min each in 50% ethanol, 30% ethanol and distilled water, stained for 6 min with 0.50% pyroninY and 0.30% methyl green in distilled water. Slides are washed twice for 20 min and 3 min in Nbutyl alcohol, and then cleared for at least 10 min each in two changes of xylene. The slides are removed from the xylene one at a time, two drops of a mounting fixative (Permount, Fisher Scientific, Pittsburgh, PA) are added to the smears, and a coverslip is placed over the slide. It is important not to remove all the slides at once from the xylene because the smears will dry resulting in a white coating over the smear, making the cells indistinguishable. Any air bubbles trapped under the coverslip are pressed out with a pair of forceps. When the Permount dries, store the slides in the dark to prevent oxidation of the stain until DMSCC are performed. Only cells that stain positive for methyl green, or positive staining for DNA, are counted. Anucleated cytoplasmic particles will stain red. Factors Influencing Cell Counts in Milk Goats. Intramammary infection is a significant cause of increased goat MSCC (Table 1). The MSCC geometric mean for halves without IMI was 672 103 cell/ml and the arithmetic mean was 675 103 cell/ml. For infected halves, geometric mean MSCC averaged 1641 103/ml and when reported as arithmetic means, 2076 103/ml. The MSCC varies, depending on the pathogen infecting the mammary gland. Infections by bacteria classified as major pathogens (Staphylococcus aureus, gram-negative bacilli [Escherichia coli and Pseudomonas spp.], Mycoplasma spp., Proteus spp., Arcanobacterium pyogenes, and Streptococci spp.) result in higher MSCC (Poutrel et al., 1996) than infections caused by minor pathogens such as coagulasenegative staphylococci (CNS). In the absence of infections by major pathogens, infections by minor pathogens are important contributors to the total bulk tank MSCC. In addition, CNS are capable of persisting in the caprine udder as a subclinical infection and have the capacity to cause clinical mastitis. Among CNS there is an association between novobiocin resistance and MSCC (Deinhofer, 1993). Novobiocin-resistant strains cause slight increases in MSCC, whereas strains sensitive to novobiocin cause large increases in MSCC.
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Intramammary infections by mycoplasma contribute significantly to increased MSCC in goats. Mycoplasmoses are diseases affecting small ruminants around the world, and endemically in the Mediterranean Basin (contagious agalactia). Mycoplasma agalactiae, several Mycoplasma spp. (such as Mycoplasma mycoides, Mycoplasma capricolum, and Mycoplasma putrefaciens) cause IMI in goats (Vergonier et al., 1997), whereas ewes are only infected by Mycoplasma agalactiae. Clinical outbreaks of contagious agalactia in small ruminants are responsible for large increases in bulk tank MSCC (Luengo et al., 1998). Viral infection by caprine arthritis encephalitis virus (CAEV) has also been reported to increase MSCC (Ryan et al., 1993). The mammary gland is one of the targets for this virus that infects macrophages. Goats infected with CAEV can show signs of clinical mastitis, but most infections by CAEV are nonclinical. For halves free of IMI by bacteria, CAEV and increased MSCC are associated, but the increase in MSCC is small, and some investigators have compared it to the increase in MSCC caused by CNS. On the other hand, little is know about the effect of CAEV on mammary glands infected by bacteria. Some results suggest that the MSCC response to intramammary pathogens is lower when goats are infected by CAEV. This may be related to reduced recruitment of leukocytes to the gland due to reduced secretion of cytokines by macrophages infected with CAEV. Noninfectious factors such as parity, stage of lactation, season and milk yield also have been related to increased goat MSCC (Paape and Capuco, 1997; Paape and Contreras, 1997; Sanchez et al., 1998). In one study, it was determined that more than 90% of the variation in MSCC in goats was not due to IMI (Wilson et al., 1995). Increasing DIM and month of the year were among the most important factors contributing to increased cell count in the absence of IMI. To a lesser extent, parity and reduced milk production also contributed significantly to increased cell count. Interestingly, 75% of the variation was unexplained. In Murciano-Granadina goats free of CAEV and IMI, a progressive increase was observed in geometric mean MSCC from first (140 103 cell/ml) to fifth lactation (600 103 cell/ml) (Sanchez et al., 1998). The increase in MSCC for stage of lactation, season, and milk yield were associated with increasing parity. Most authors point out that the increase in MSCC throughout lactation could be explained by a dilution effect, because milk production decreases with increasing stage of lactation, and MSCC follows a linear increase throughout lactation. In addition, when the stage of lactation is similar, the effect of season on MSCC was shown to be related to milk production (Sanchez et al., 1998). Other factors reported to increase MSCC in goats are estrus, vaccination, change in diet and change in the milking routine (Paape and Contreras, 1997). Because these factors result in physiological stress, to which goats are very sensitive, the resulting decrease in milk yield could explain the increase in MSCC. Ewes. Various noninfectious factors have been associated with increased cell counts in sheep milk. The most significant are parity, stage of lactation, season, herd, handling of ewes, and diurnal variation (Gonzalo et al., 1994, 1995; Gonzalo and San Primitivo, 1998). For uninfected mammary glands, MSCC are highest on the day of parturition (596 103 cells/ml) and decrease during the transition from colostrum to true milk, averaging 239 103 and 186 103/ml at 5 and 12 d of lactaE240 Journal of Dairy Science

Table 3. Monthly milk SCC (cells 103/ml) throughout lactation in half udders of ewes.1 Infection status2 Month of lactation |n
3

6 0394 1148 2108

8 0477 1999 4272

Mean 0185 0576 1445

Negative 579 0145 0088 0118 Recurring 535 0311 0247 0416 Positive 92 1426 1101 1226 1 Adapted from Romeo et al. (1996). 2 Bacteriogical status throughout lactation. 3 Number of halves.

tion (Table 3). Counts continue to decrease with increasing milk production and reach minimum values of approximately 30 103/ml at the fifth wk of lactation, which coincides with maximum milk production. Counts remain unchanged for the remainder of the lactation. The number of lambs delivered at lambing does not influence the MSCC (Gonzalo et al., 1994). For the majority of milk-producing breeds, there is seasonal breeding with most ewes lambing in the winter months. Thus, the seasonal increases in MSCC are linked to the normal lactation curves, where milk production is lowest during summer and winter months. A 4 to 11% increase in MSCC occur between the first and fourth lactation. The diurnal and daily variations between milkings are similar to those observed in dairy cows. A 70% increase in the MSCC was observed 1 h after milking (Gonzalo et al., 1994). Other nonsystematic factors contributing to variation in MSCC such as changes in feeding have not been studied. The geometric mean MSCC of uninfected mammary glands is <100 103/ml, very similar to that of dairy cows (GonzalezRodriguez et al., 1995; Marco, 1996; Romeo et al., 1996). The geometric mean MSCC for mammary glands infected with Micrococcus, corynebacteria, and yeasts is between 100 103 and 500 103 cells/ml. Milk from halves infected with CNS has a highly variable MSCC. Infection by those strains resistant to novobiocin (Staphylococcus xylosus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus gallinarum, Staphylococcus gallinarum and Staphylococcus lentus) cause an average MSCC of 200 103 cells/ml, while infection by CNS sensitive to novobiocine (Staphylococcus simulans, Staphylococcus epidermis, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus caprae) counts average more than 1 106 cells/ml. Staphylococcus simulans produce greater increases in MSCC than other species of CNS and induce clinical symptoms similar to infections by major classical pathogens (Bergonier et al., 1996; GonzalezRodriguez et al., 1995; Marco, 1996). Ewes have a greater cell response to subclinical IMI than dairy cows. In ewes, the MSCC can reach counts of 20, 40, and even 60 106/ml without clinical symptoms and producing milk of normal macroscopic appearance (Gonzalo and San Primitivo, 1998; Green, 1984). There is conflicting information with respect to the influence of IMI by the Maedi/Visna (M/V) virus on MSCC. However, in one study carried out on a herd of 82 ewes with a high prevalence of infection by M/V (39.1%), the results indicated that although bacterial infection was the major factor contributing to elevated MSCC, seropositivity against M/V was significantly associated with MSCC (Gonzalo and San Primitivo, 1998). The least-square means of log10 MSCC were 5.52 0.08, 5.77 0.09, and 5.97 0.10, respectively, for ewes with negative, weakly positive, and strongly positive

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Table 4. Percentage of producers in relation to bulk tank milk SCC.1 SCC 10 /ml Cooperative2 < 0.75 0.75 to 1 1 to 2 >2
6

Table 5. Effect of milk SCC (MSCC) on milk yield and composition (n = 20,796 goats).1 MSCC 103/ml Parameter < 750 7501750 Yield (kg) 771a 710b Fat (g/kg) 31.9a 31.6b a 29.1b Protein (g/kg) 28.6 a,b,c Means in a row with different superscripts differ (P < 0.05). 1 Adapted from Baudry et al. (1997). >1750 641c 31.6b 30.1c

--------------------------------%---------------------------------1 05.0 25.0 60.0 10.0 2 11.0 28.0 44.0 17.0 3 07.0 07.0 43.0 43.0 4 00 22.0 78.0 00 5 20.0 40.0 40.0 00 Combined data 08.6 25.9 51.9 13.6 1 Adapted from Droke et al. (1993). 2 Seventy one dairy goat herds among the 5 dairy cooperatives.

negative, weakly positive, and strongly positive serology. From studies carried out in meat breeds, a MSCC for uinfected ewes was approximately 1 106/ml (Fthenakis, 1996; Green, 1984; Maisi et al., 1987). Later studies conducted on both meat and dairy Spanish breeds established a threshold MSCC at 250 103 cells/ml for discriminating uninfected from infected mammary glands. The MSCC for discriminating between healthy and infected glands are similar to those described for cattle at 200 to 400 103 cells/ml. At these levels, the percentage of samples correctly classified as infected or uninfected is between 71 and 85%. The sensitivity and specificity values were between 71 and 95%, and the positive and negative predictive values were between 65 and 95% (Feltran de Heredia et al., 1988; Gonzalez-Rodrigues et al., 1995; Marco, 1996). Significance of Somatic Cells in Milk Some researchers feel that the MSCC is not an appropriate method for predicting mastitis in goats because of the naturally high MSCC in healthy goat halves and because noninfectious factors increase MSCC (Haenlein and Hinckley, 1995). Although there are difficulties in interpreting MSCC results for goats, counts from flouro-optic-electronic cell counters and CMT scores can be used; but their predictive values will be lower than in other domestic ruminants. Different MSCC thresholds have been proposed to diagnose infected halves, but the thresholds are always higher than those proposed for cows. Poutrel and Lerondelle (1983) found that a threshold MSCC of 1 106 cells/ml would correctly classify 85% of IMI by major pathogens, but the percentage of correctly classified IMI decreased when all pathogens, including CNS, were included. The use of a dynamic threshold that included stage of lactation period and other factors has recently been proposed (Sanchez et al., 1998). For CMT results, the first three scores (negative, trace, and 1) should be avoided and only scores 2 and greater should be used to predict IMI. Age, stage of lactation, and other noninfectious factors that increase MSCC should be considered when interpreting CMT results. The predictive value for a negative CMT score is high (87% uninfected), but the predictive value for a positive test score of 2 and greater is low (34% infected) (Contreras et al., 1996). Thus, the CMT could be useful in avoiding healthy udder halves when using selective antibiotic therapy at drying off. In the United States the legal limit for sheep milk is 0.75 106 cells/ml and for goat milk it is 1 106 cells/ml (Pasteurized Milk Ordinance, 1995). Official rules for establishing a legal MSCC limit for bulk tank milk have not yet been established in the EU for small ruminants. However, this

situation is only temporary because the EU has suggested establishing a future legal limit for bulk tank milk (European Economic Community/92/46 Directive). In France, a quality premium payment system for goat milk was recommended by the French Association of goat milk producers and processors that should be officially recognized in the year 2000 (Charpentier, 2000). The recommended quality premium payment system will include the following criteria: total bacterial counts, MSCC, IgG1 (an indicator of the presence of colostrum in bulk tank milk), antibiotics, and lipolysis. In the United States, a legal limit of 1 106 cells/ml of milk was established for goat milk and many dairy goat farmers frequently cannot meet this limit. Droke et al. (1993) reported that 34.5% of the producers were in compliance with the current goat MSCC standard of 1 106 cells/ml, and only 8.6% had MSCC <0.75 106/ml, which is the current cow MSCC in the US (Table 4). At the International Symposium of Somatic Cells and Milk of Small Ruminants (1994), it was suggested to the EU authorities that a legal SCC limit for bulk tank milk for goats and sheep should not be lower than 1.5 106 cells/ml. A study to determine the effect of these MSCC limits on the number of goat producers that were unable to meet the MSCC limit were carried out in Murcia, Spain (Luengo et al., unpublished). It was concluded that the US limit would be too strict and that seasonal variation needed to be considered. With the US limit, 20% of the herds would have their milk rejected for the entire lactation. The suggested limit of 1.5 106 cells/ml was more favorable because most herds were below that threshold. Changes also occur in milk yield and composition of goat milk with increasing MSCC (Baudry et al., 1997). Milk yield and the yield (g/kg of milk) of fat decreased and total protein increased with increasing cell count (Table 5). The increase in protein was attributed to an increase in bovine serum albumin attributed to a breakdown of the blood milk barrier caused by inflammation of the udder. A decrease in milk yield with increasing MSCC was also reported in sheep (Gonzalo et al., 1994). Most, if not all, of the negative effects of elevated MSCC on milk yield and composition is caused by the normal function of PMN (Akers and Thompson, 1987; Capuco et al., 1986; Paape et al., 1995). Activated PMN recruited to the gland release several radical species, including superoxide anion and hydrogen peroxide. Superoxide anion and nitric oxide react to form peroxynitrite (Beckman et al., 1990), a strong oxidant that has detrimental effects on epithelial cells. Peroxynitrite is capable of crossing cell membranes, oxidizes DNA, interrupts mitochondrial respiration, modifies proteins, including the nitration of tyrosine residues, and induces cell death. Increased myeloperoxidase in milk, released by PMN, is highly correlated with MSCC (Cooray, 1994). In the presence of hydrogen peroxide both myeloperoxidase and lactoperoxidase oxidize nitrite to nitrogen dioxide, which cross links
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Table 6. Experimental design used to study effects of postmilking teat dipping on new infection rates and milk SCC (MSSC).1 Trial 1 No. of herds No. of goats Active ingredient Criteria 6 dipped 6 undipped 1001 Nisin Trial 2 12 dipped 245 undipped 21,331 Iodine Chlorhexidine MSCC Table 8. Experimental design used to test efficacy of administration of antibiotics at dry-off.1 Trial 1 Trial 2 5 12 No. of herds No. of goats 215 1000 Product NAFPENZAL ORBENIN ND DC (Intervet) (Pfizer) Active ingredient Benzylpenicillin Cloxacillin Nafcillin No. of bacteriological analysis 860 3112 1 Adapted from Poutrel et al. (unpublished data).

Bacteriology d 0 to 7 and mo 7 to 10, n = 3874 1 Adapted from Poutrel et al. (unpublished data).

proteins, oxidizes cysteine residues, and nitrates tyrosine residues (Van der Vliet et al., 1997). Efficacy of Postmilking Teat Dipping and Intramammary Administration of Antibiotics at Dry-off Teat dipping. Postmilking teat dipping has been shown to be effective in reducing new intamammary infection in dairy cows (National Mastitis Council, 1996). Poutrel et al. (unpublished data) recently reported on the effects of postmilking teat dipping on new IMI rate and bulk milk MSCC in goats. Two trials were conducted on 3001 goats (Table 6). In trial one, nisin was the active ingredient in the teat dip, and in trial two teat dips containing either iodine or chlorhexidine were used. The teat dip was applied using teat dip cups. For trial one, the rate of new intramammary infection averaged 29.2 and 50.9% (P < 0.01) for nisin dipped and undipped udder halves (Table 7). For trial two, bulk milk SCC averaged 968 and 1420 103/ml for the iodine or chlorhexidine dipped and undipped treatments (Table 7). These data indicate the beneficial effect that postmilking teat dipping had on new IMI rate and MSCC. For that study 2.3 l of teat dip was used/goat/year. For herds using the teat dip, an additional milking time of 10 to 15 m/herd was necessary. Intramammary administration of antibiotics at dry-off. Intramammary administration of antibiotics at dry-off to dairy cows is routinely used to reduce existing and new IMI (National Mastitis Council, 1996). Studies with goats have reported high cure rates (72.5 to 79%) of infected halves after antibiotic therapy at dry-off (Fox et al., 1992; Luengo et al, 2000; Poutrel et al., 1997). Similar cure rates have been reported for ewes (Longo et al., 1996; Marco, 1996; Tardaguila et al., 1997). The spontaneous cure rate during the dry period for goats averaged 20 to 50% (Poutrel et al., 1997) and for ewes 20 to 56% (Corrales et al. 1996). All authors agree that treatment with antibiotics at dry-off had no effect on reducing new IMI in goats and ewes. At the 7th International Conference on Goats held May 15 to 18, 2000, in Tours,
Table 7. Effect of postmilking teat dipping with nisin on new IMI and milk SCC (MSCC).1 Trial 1 Treatment No. of goats New infections2 Trial 2 MSCC 103/ml

France, the practice of selective or systematic treatment was discussed. From that discussion, it was concluded that selective treatment of infected goat mammary halves should be practiced, and that systematic dry therapy should be used only in goat herds with a high prevalence of subclinical mastitis (>30 to 40%). A decrease in bulk tank MSCC was observed after kidding for herds practicing dry-off therapy when coupled with milking hygiene and use of post-milking teat dips. In one study (Luengo et al., 2000) on eight herds for two consecutive lactations, reduced bulk tank MSCC was observed over the entire lactation following dry-off therapy compared with the previous lactation for six of the herds (Figure 2). Selective dry treatment was used for six herds and systematic treatment for two herds. All herds practiced teat dipping postmilking. Two unpublished trials were conducted in goats to test efficacy of administration of antibiotics at dryoff (Poutrel et al., unpublished data). In trial one, 215 goats received an intramammary infusion of a product formulated for dairy cows containing benzylpenicillin, dihydrostreptomycin, and nafcillin (Table 8). In trial two, 1000 goats received a product formulated for dairy cows containing cloxacillin as the active ingredient. The amount infused was the same as that administered to dairy cows at dry-off. Both treatments resulted in an increase in the percentage of uninfected quarters at kidding when compared to the percentage at dry-off (Table 9). In trial one, the percentage of uninfected halves increased from 63% at dry-off to 91% at kidding. In trial two, the percentage of uninfected halves

1400 1200 MSCC x 10 /ml 1000 800 600 400 200 0 1 2 3 4 5 Herd
Figure 2. Effect of selective or systematic treatment with antibiotics at dry-off on average bulk tank milk SCC (MSCC) for eight herds for two consecutive lactations. Herds 1 to 6 received selective antibiotic treatment, herds 7 and 8 received systematic treatment. Light bars = lactations during 1997 to 1998; dark bars = lactations during 1998 to 1999. *Significantly lower (P = 0.0001) than MSCC in lactation 1997 to 1998. Adapted from Luengo et al. (unpublished data).
3

* *

* * * *

8 Avg.

Dipped 241 29.2 0968 Undipped 381 50.9 1420 P <0.01 <0.001 1 Adapted from Poutrel et al. (unpublished data). 2 New infections 100/total no. of udder halves initially uninfected. E242 Journal of Dairy Science

PAAPE ET AL.
Table 9. Efficacy of intramammary administration of antibiotics at dryoff.1 Trial 1 Trial 2 Infection status Uninfected Dry-off No. % 269 63 Kidding No. % 391 91 1 8 Dry-off No. % 840 55 111 604 7 39 Kidding No. % 131 85 9 53 3 184 12

REFERENCES
Akers, R. M., and W. Thompson. 1987. Effect of induced leucocyte migration on mammary cell morphology and milk component biosynthesis. J. Dairy Sci. 70:1685-1695. Baudry, C. R., R. de Cremoux, C. Chartier, and G. Perrin. 1997. Incidence de la concentration cellulaire du lait de chvre sur sa production et sa composition. Vet. Res. 28:277-286. Barbosa, M., F. Barillet, X. Berthelot, S. Casu, A. Foglini, A. D. Gabi-A, G. Kalantzopoulos, A. Ledda, G. Perrin, B. Poutrel, J. Renaud, and R. Rubino. 1994. Conclusioni delcomitato Scientfico International Symposium Somatic Cells and Milk of Small Ruminants, Italia. Beckman, J. S., T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman. 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA. 87:1620-1624. Beltrn de Heredia, F., and J. Iturritza. 1988. Recuento de clulas somticas en leche de ovejas de raza Latxa. II Determinacin del umbral fisiolgico. Medical Vet. 5:33-38. Bergonier, D., F. Longo, G. Lagriffoul, P. J. Consalvi, A. Van de Wiele, and X. Berthelot. 1996. Frquence et persistance des staphylocoques coagulase ngative au tarissement et relations avec les numrations cellulaires chez la brebis laitire. Pages 53-59 in Somatic Cells and Milk of Small Ruminants. R. Rubino, ed. Wageningen, the Netherlands. Bergonier, D., X. Berthelot, and F. Poumarat. 1997. Contagious agalactia of small ruminants:current knowledge concerning epidemiology, diagnosis and control. Rev. Sci. Tech. Office Int. des Epizooties. 16:848-873. Capuco, A. V., M. J. Paape, and S. C. Nickerson. 1986. In vitro study of polymorphonuclear leukocyte damage to mammary tissues of lactating cows. Am. J. Vet. Res. 47:663-668. Carroll, E. J., and O. W. Schalm. 1962. Effect of deoxyribonuclease on the California test for mastitis. J. Dairy Sci. 45:1094-1097. Charpentier, P. 2000. Quality payment system for goats milk: Recommendations of the French association of goats milk producers and processors. Pages 761-763 in Proc. Int. Conf. on Goats. May 15-18, Tours, France. Contreras, A., D. Sierra, J. C. Corrales, A. Snchez, and J. Marco. 1996. Physiological threshold of somatic-cell count and California Mastitis Test for diagnosis of caprine subclinical mastitis. Small Ruminant Res. 21:259-264. Contreras, A., M. J. Paape, A. L. DiCarlo, R. H. Miller, and P. Rainard. 1997. Evaluation of selected antibiotic residue screening test for milk from individual goats. J. Dairy Sci. 80:1113-1119. Contreras, A., D. Sierra, J. C. Corrales, A. Sanchez, and C. Gonzalo. 1998. Diagnostico indirecto de las mamitis caprinas. Mamitis caprina II. Ovis. 54:25-36. Cooray, R. 1994. Use of bovine myeloperoxidase as an indicator of mastitis in dairy cattle. Vet. Micro. 42:317-326. Corlett, N. J., M. J. Paape, T. K. Ledbetter, M. E. Bowman, and B. D. Rhoads. 2000. Preparation of high range milk somatic cell count standards for calibration of electronic instruments. Pages 162-163 in Proc. 39th Annu. Mtg. Natl. Mastitis Counc., Madison, WI. Corrales, J. C., A. Snchez, D. Sierra, J. C. Marco, and A. Contreras. 1996. Relationship between somatic cell counts and intramammary pathogens goats. Pages 35-39 in Somatic Cells and Milk of Small Ruminants. R. Rubino ed. Wageningen, the Netherlands. Corrales, J. C., D. Sierra, A. Sanchez, and A. Contreras. 1994. Control de la eficacia del tratamiento de secado en cabras murciano-granadina. Pages 303-308 in Proc. XIX J. de la Sociedad Espaola de Ovinotecnia y Caprinotecnia. Burgos, Spain. Cuccuru, C., P. Moroni, A. Zecconi, S. Casu, A. Caria and A. Contini. 1997. Milk differential cell counts in relation to total counts in Sardinian ewes. Small Ruminant Res. 25:169-173. De Cremoux, R. B. Poutrel, R. Pillet, G. Perrin, M. Ducellier, and V. Heuchel. 1996. Cell counts for diagnosing caprine bacterial mammary infections. Pages 35-39 in Somatic Cells and Milk of Small Ruminants. R. Rubino ed. Wageningen, the Netherlands. Deinhofer, M. 1993. Staphylococcus spp. as mastitis-related pathogen in ewes and goats. Pages 136-143 in Proc. 5th Int. Symp. Machine Milking of Small Ruminants. De la Cruz, M. E. Serrano, V. Montoro, J. C. Marco, M. Romeo, R. Baselga, I. Albizu, and B. Amorena. 1994. Etiology and prevalence of subclinical mastitis in the Manchega sheep at mid-late lactation. Small Ruminant Res. 14:175-180. Droke, E. A., M. J. Paape, and A. L. Di Carlo. 1993. Prevalence of high somatic cell counts in bulk tank goat milk. J. Dairy Sci. 76:1035-1039. Dulin, A. M., M. J. Paape, and W. P. Wergin. 1982. Differentiation and enumeration of somatic cells in goat milk. J. Food Prot. 45:435-439. Dulin, A. M., M. J. Paape, W. D. Schultze, and B. T. Weinland. 1983. Effect of parity, stage of lactation, and intramammary infection on concentration of Vol. 84, E. Suppl., 2001 E243

Major patho3 1 4 gens Minor patho157 37 34 gens 1 Adapted from Poutrel et al. (2000).

increased from 55% at dry-off to 85% at kidding. A decrease in the percentage of udder halves infected by major and minor pathogens was observed. In another study by the same authors it was shown that failure to teat dip after kidding nullified the beneficial effect of dry treatment (Table 10). Based on MSCC of control and antibiotic-treated udder halves, counts averaged 546 and 270 103/ml for control and treated halves at 50 to 75 d after kidding (P < 0.05). However, by 75 to 100 d after kidding MSCC for both groups were similar (P > 0.05). The authors concluded that teat dipping after kidding was necessary to maintain the beneficial effect obtained by antibiotic therapy at dry-off. For all of the trials, no detectable antibiotic residue was found at kidding.
CONCLUSIONS

The MSCC from uninfected mammary glands of ewes are similar to counts in cow milk, averaging less than 100 103/ml. For uninfected goat mammary glands, MSCC are higher and can approach 2 106/ml. The macrophage is the predominant cell type in uninfected milk from ewes, whereas the PMN is the predominant cell type in goats. Milk secretion in goats and ewes is primarily apocrine in nature. This results in the shedding of numerous anucleated cytoplasmic particles from the apical surface of mammary secretory cells during milk secretion. Because of the larger number and size of cytoplasmic particles secreted in goat milk compared with ewes and cows, only counting methods that can distinguish these cell-like cytoplasmic particles from somatic cells should be used for evaluating goat milk. Intramammary infection is the major cause for increased MSCC in goats and ewes. However, noninfectious factors such as parity, stage of lactation, season, and milk yield have been related to increased MSCC for goats and ewes. As a result, North American goat dairymen have difficulty maintaining MSCC below the legal limit of 1 106/ml. Increased cell counts in goat and sheep milk is associated with reduced milk and fat yields. Intramammary administration of antibiotics to goats at dry-off and postmilking teat dipping after kidding will reduce the incidence of new IMI and lower MSCC.
Table 10. Lack of teat dipping after kidding nullifies effect of dry treatment.1 Time Control Treated Significance (SCC 103/ml) 0923 NS 1204 NS P < 0.05 NS

Before dry-off -75 to -50 d 933 -50 to -25 d 925 After kidding + 50 to + 75 d 546 0270 + 75 to + 100 d 579 0544 1 Adapted from Poutrel et al. (unpublished data).

SYMPOSIUM: SMALL RUMINANT DAIRY RESEARCH AND PRODUCTION

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