Relationship between mammary gland infections and some milk immune parameters in Sardinian breed ewes

Small Ruminant Research 41 (2001) 1±7

Relationship between mammary gland infections and some milk immune parameters in Sardinian breed ewes P. Moronia,*, C. Cuccurub a

Dipartimento di Patologia Animale, Igiene e SanitaÁ Pubblica Veterinaria, UniversitaÁ degli Studi di Milano, via Celoria 10, 20133 Milano, Italy b Istituto di Patologia Speciale e Clinica Medica, UniversitaÁ degli Studi di Sassari, Italy Accepted 25 March 2001

Abstract Over the last few years an increased rate of intramammary infections caused by environmental and opportunistic pathogens has been observed in sheep farms. The presence of these microorganisms is mainly related to poor conditions of environmental hygiene and/or to decreased defenses of the mammary gland. The work we present here is a part of a project on the immune status of ewe mammary gland and its in¯uence on mastitis development. In this paper, we have studied how intramammary infections caused by opportunistic pathogens re¯ect on immune parameters in ewe milk. Milk samples collected monthly before the morning milking, were screened for the presence of microorganisms and tested for somatic cell counts (SCC), polymorphonuclear neutrophil leukocytes (PMN), phagocytic activity, lysozyme content, and NAGase activity. Data showed that phagocytic activity was signi®cantly higher in bacteriologically negative udders (156 mV/1000 PMNs) than in infected udders (10 mV/1000 PMNs). These results suggest that intramammary infection might be associated with a decrease of mammary gland immune status. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Ewes; Mammary infections; Immune parameters

1. Introduction Sanitary procedures and programs can be considered as the most effective means to prevent or control mastitis and to obtain high quality milk with low somatic cell counts (SCC). Somatic cell counts represent the intensity of the cellular immune defenses in response to an in¯ammatory process and in cows SCC are widely used as a marker to establish udder health *

Corresponding author. Tel.: ‡39-2-70638626; fax: ‡39-2-70635338. E-mail address: [email protected] (P. Moroni).

and milk quality (Paape et al., 1984). Cow and small ruminant breeding farms are quite different in environment and management conditions and the target of high quality milk production is perhaps more challenging in small ruminants than in cows. Over the last years the progress in ewe breeding, and in particular the introduction of sanitary programs and milking machines, led to a reduction of infections caused by Staphylococcus aureus and conversely to a parallel increase of coagulase negative staphylococci (CNS) (Cuccuru et al., 1996). Mammary gland immune defenses play an important role in the prevention of mammary infections and

0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 1 9 3 - 6

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P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7

the reduction of local immunity usually coincides with an increase in clinical cases of mastitis (Lee and Outteridge, 1981). While in the literature on dairy cows there is a general consensus about the importance of creating a model to evaluate mammary gland immune status, in small ruminants this aspect has been scarcely studied. Such a model should take into consideration milk components (cells, enzymes, and proteins) and all the factors that might determine variations of these components (management, age of animals, number of lactations, nutrition, milking system). The understanding of the mechanisms that regulate immunity in small ruminants would be helpful to clarify the relation between intramammary infections and immune defenses allowing the development of an ef®cient program for the prevention of mastitis. In this work, we have studied in 40 Sardinian ewes the correlation between mammary gland infections and some milk parameters representing the status of mammary gland immunity (somatic cell counts, the number and the phagocytic activity of PMNs and humoral immunity (lysozyme content and NAGase activity).

and no contagious microorganisms were isolated from the milk either before or over the experimentation.

2. Materials and methods

2.4. Microbiological assay

2.1. Study design

Bacteriological screening was carried on following NMC procedures (NMC, 1987). Brie¯y 10 ml of each sample was spread onto the surface of 5% bovine blood agar plates and incubated at 378C for 18±24 h. No contagious microorganisms (Streptococcus agalactiae and Staphylococcus aureus) were isolated. A teat was de®ned as infected when >1000 UFC/ml (FIL-IDF, 1981) of environmental pathogen streptococci (other than S. agalactiae) or staphylococci (other than S. aureus) were found. In all other cases, the samples were considered uninfected. We found only coagulase negative staphylococci (CNS) and those were classi®ed using standard microbiological procedures and the API System (Bio Merieux Italy, Rome).

A complete lactation follow-up study was conducted on 40 Sardinian ewes belonging to a semiextensive herd located in the North±West of Sardinia and formed by 198 ewes in lactation (55 primiparous, 143 pluriparous) and seven lambs. Animals were mated in the second half of August and consequently they lambed in the last 15 days of January. During the lactation, the animals were fed on pasture, concentrate and alfalfa hay depending on milk production period. The ewes were kept together with the lambs for 30±35 days before the start of milking (March±July). After weaning, all ewes were milked twice a day with milking machine without teat preparation nor teat dipping after milking. Milking machine working parameters were characterised by a vacuum level of 44 kPa, 180 pulsations/min, and a 1/1 aspiration/massage rate. External and internal diameters of the teat cup were 17 and 11 mm, respectively. Animals received no therapy at the start of the dry period

2.2. Animals A total of 40 animals at the beginning of lactation were divided in two groups (healthy and infected) in relation to the status of infections of the half udders; analysis were undertaken monthly during the entire lactation. 2.3. Sampling Monthly, from April to July (T1±T2±T3±T4), 100 ml of foremilk was collected in sterile tubes just before morning milking. Milk, samples were collected after teat disinfection with chlorhexidine moistened towels and after discarding the ®rst milk streams. Tubes were cooled to 48C and transferred to the laboratory for microbiological and cytological analysis within 1±2 h after collection. For SCC evaluation, an additional 10±15 ml of milk was collected in large tubes, already containing 0.25 ml of bronopol, and then stored at 48C.

2.5. Somatic cell conts The number of somatic cells was estimated using the ¯uoro-opto-electronic method (Fossomatic, Este, Italy) following FIL-IDF (1995) recommendations.

P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7

2.6. Differential somatic cell counts Milk cells were isolated using density gradient centrifugation and cell suspensions were estimated by esterase staining (Yam et al., 1971). 2.7. PMNs separation A total of 100 ml of milk were centrifuged for 15 min at 400  g at room temperature; after removing the supernatant, the cell pellets were resuspended in 4 ml of Hank's balanced salts solution (HBSS) without Ca2‡ and Mg2‡ (Sigma, 20100 Milan, Italy) and carefully transferred into 15 ml conical centrifuge tubes containing 3 ml Ficoll±Paque (Pharmacia Biotech, Uppsala, Sweden). After centrifugation at 400  g for 30 min at room temperature, the supernatants were removed and cell pellets were washed twice with 10 ml HBSS by centrifuging at 400  g for 10 min at room temperature. Cells were then resuspended again in 1 ml HBSS added with 10% glucose and 5% albumin and stored at 48C. Cells viability was estimated by the Trypan blue exclusion method. 2.8. Preparation of stimuli and opsonisation Zymosan was prepared following the procedures of Metcalf et al. (1986). After incubation for 30 min at 378C PMN opsonisation was obtained by adding 2 ml zymosan suspension to the same quantity of whey derived from pooled milk of healthy ewes different from those used in the trial. Opsonised suspension was immediately used for phagocytosis assay. 2.9. Phagocytic activity assay Luminol chemiluminescence was used to determine milk PMNs phagocytic activity (Dulin et al., 1988). A total of 100 ml of cells suspension (PMN) was added to 100 ml of opsonised suspension, which had been diluted 1:10 with HBSS. A total of 800 ml of a 10 4 M luminol solution (Bio-Orbit) was added to the suspension. Samples were assessed with a luminometer LKB Wallac 1250 (LKB Italy). Values were recorded for 30 min. Data were stored by software (Bio-Orbit) that recorded mV luminometric response. Values were ®nally analysed by instrumental curve

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analysis software (Peak ®t, Jandel, Germany) calculating the area under the curve. 2.10. NAGase Milk whey NAGase activity was measured as described by Kitchen et al. (1978). It evaluated the ¯uorescence emitted by 4-methylumbelliferone during the reaction (at 378C and pH 4.6) between the 4methylumbelliferyl-N-acetyl-b-glucosaminide (SIGMA, Milan, Italy) substrate with the NAGase contained in the analysed samples. Samples were assessed after calibration of the instrument with 4methylumbelliferone graduate solutions. Fluorescence was read with a ¯uorimeter (Kontron Instruments, Milan, Italy) at the wavelength of excitation and emission, 360 and 445 nm, respectively, and data were recorded with a plotter. About 1 unit of NAGase activity represents the amount of 4-methylumbelliferone picomoles released from 1 ml of milk in 1 min at 258C. 2.11. Lysozyme Milk whey lysozyme was evaluated spectrophotometrically following Metcalf et al. (1986). This method measures the sensitivity of Micrococcus lysodeikticus (Sigma, Milan) cell wall to the lythic action of lysozyme. A total of 30 ml of 1:5 diluted milk whey samples (in phosphate buffer solution) was added to a Micrococcus lysodeikticus suspension of known density (o:d: ˆ 0:6±0.8). After 2 min incubation the drop in optical density was measured spectrophotometrically at 450 nm wavelength for triplicate. Data were analysed by linear regression using a graphic software program (Sigma Plot, Jandel, Germany). 2.12. Statistical analyses The level of signi®cance was determinated using the Chi-square (w2) test and all values with P < 0:05 were considered as signi®cant. 3. Results Udders were divided in two groups, named healthy and infected, in relation to their bacteriological status.

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P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7

Table 1 Samples distribution and prevalence of infections Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 7 8 7

20.0 18.9 23.5 23.3

32 30 26 23

80.0 81.1 76.5 76.7

Table 2 shows milk SCC in the healthy (CNS-negative) and in the infected (CNS-positive) group. Within each group, SCC remained stable for the entire experimental period. The SCC was signi®cantly higher in the infected group (6.34 Log10/ml) than in the healthy group (4.96 Log10/ml). The same trend was also observed in the number of PMNs (Table 3).

In each sampling (from T1 to T4), the number of healthy udders ranged between 7 and 8, while infected udders ranged between 22 and 32. The high percentage of infection (80%) did not allow us to obtain a homogeneous distribution of animals between the two groups. The reduction in the number of samples, especially at T3 and T4, was due to the death of four animals (three before the second sampling and one before the third sampling) and to the impossibility of collecting samples from ®ve ewes which started the dry period early. Table 1 shows the ratio between infection prevalence and sampling. The decrease in infection prevalence (80±76%) is a function of the reduction in the total number of samples rather than an improvement of sanitary conditions.

3.1. Milk whey NAGase activity Though the origin of NAGase in milk is controversial, it is generally agreed that PMNs are the major source of soluble NAGase (Dulin et al., 1984). The role of leukocytes in NAGase production is suggested by the observation of an increase of both values during clinical and subclinical mastitis (Kaar-

Table 2 Log10 milk SCC arithmetical mean trend of positive and negative samples Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 6 8 6

4.97 4.91 4.89 5.08

31 30 26 22

6.31 6.16 6.49 6.40

*

   

0.48 0.39 0.26 0.34

   

0.66* 0.61* 0.54* 0.51*

Signi®cant P < 0:05.

Table 3 PMNs mean in positive and negative samples Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 6 8 6

31.87 52.66 31.12 33.83

31 30 26 22

77.60 73.26 68.69 65.95

*

Signi®cant P < 0:05.

   

15.86 23.69 16.28 10.30

   

16.82* 19.86* 18.41* 19.85*

P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7

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Table 4 Milk whey NAGase (U/ml) activity in positive and negative samples Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 7 8 7

15.62 11.14 7.75 20.28

32 30 26 23

24.75 27.76 33.11 45.30

*

   

14.96 5.08 7.06 4.53

   

14.83 18.44* 18.32* 22.94*

Signi®cant P < 0:05.

showed the highest concentration at T1, even though it was approximately double in negative (512 mg/ml) than in positive (243 mg/ml) samples. Although in both positive and negative groups whey lysozyme concentrations were lower at T2, T3, and T4 compared to T1, in positive samples it remained fairly stable over the three months, whereas in the negative samples the concentration showed a 10 times drop at T2 followed by a progressive increase at T3 and T4. Concentration of lysozyme at T1, T2, T4 showed statistically signi®cant differences between the positive and negative group (Zecconi et al., 1998).

tinen et al., 1990). In¯ammatory processes increase the release of the enzyme by damaged cells and stimulate the recruitment of PMNs. Table 4 shows the result of NAGase activity in ewe milk's whey. In cows, NAGase concentration in milk whey has been reported to be higher during the early lactation and the drying-off period when compared to midlactation (Miller et al., 1990). Moreover, it has been shown that NAGase activity in bovine milk whey is also in¯uenced by the occurrence of infections in the mammary gland (Zecconi et al., 1998). Similarly, we found that in ewe whey samples collected from CNSpositive udders NAGase activity was higher than samples collected from CNS-negative udders although in both negative and positive samples NAGase activity was higher in late lactation (20.28 and 45.30 U/ml, respectively) than in early lactation (15.62 and 24.75 U/ml, respectively).

3.3. Milk PMNs phagocytc activity Table 6 are summarised the results of PMN phagocytic activity assessed in ewe milk by luminol chemiluminescence. Negative samples always showed a higher PMNs phagocytic activity in comparison to positive ones. Values in the negative group dramatically increased from T1 (3.69 mV/1000 PMN) to T4 (517.07 mV/1000 PMN). Statistically signi®cant differences between the healthy and infected groups which were found only at T3 (75.12 mV/1000 PMN versus 4.89 mV/1000 PMN) and at T4 (517.07 mV/1000 PMN versus 6.25 mV/ 1000 PMN).

3.2. Milk whey lysozyme The pattern of bovine whey lysozyme concentration has been investigated recently and showed that the level of lysozyme was higher for the ®rst 60 days after calving (Zecconi et al., 1998). Table 5 shows the pattern of lysozyme concentration in ewe whey. As in cows, ewe whey lysozyme

Table 5 Milk whey lysozyme contents (mg/ml) in positive and negative samples Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 7 8 7

512.50 51.85 99.00 191.28

32 30 26 23

243.15 102.00 94.42 103.37

*

Signi®cant P < 0:05.

   

307.16 42.32 85.76 38.94

   

145.88* 33.32* 50.88 71.04*

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P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7

Table 6 Milk PMNs phagocytic activity (mV/1000 PMN) in positive and negative samples Sampling

Samples (n)

Negative (%)

Samples (n)

Positive (%)

T1 T2 T3 T4

8 7 8 7

3.69 30.20 75.12 517.07

32 28 26 23

3.49 25.27 4.89 6.25

*

   

9.89 38.28 67.65 100.55

   

5.35 115.62 7.96* 14.75*

Signi®cant P < 0:05.

4. Conclusions Ours results con®rm the importance of SCC for the diagnosis of intramammary infections (IMI) showing very high counts in the samples of infected half udders. The high values of cells in infected half udders can also be exploited to detect IMI from foremilk. However, in the diagnosis of IMI physiological variations in SCC should be taken into account. It has been reported that above all somatic cell types in ewe milk, PMNs are the main one which tends to increase after lambing and before the dry period (Fruganti et al., 1985). The latter case is probably the result of a reduction of milk production and a contemporary physiological increase of cells protecting the gland during the dry period. Our data con®rm the high correlation between IMI high SCC and PMNs percentage that we have previously reported (Cuccuru et al., 1997). The immune status of ewe mammary gland is very important to improve environmental control and prophylaxis programs. Intramammary infections depend on extrinsic factors (such as climate, stalling type, bedding condition, etc.) and intrinsic factors (age, lactation number, lactation period, mammary gland immunity). A high number of CNS infections was observed during the whole lactation (78%) con®rming that these bacteria are the most frequent cause of mammary gland infections in ewes (White and Hinckley, 1999; Kirk et al., 1996). These infections in¯uenced by breeding management and in particular by sanitary conditions and hygienic procedures adopted before, during and after milking (Bergonier et al., 1994). The NAGase activity results are highly signi®cant to identify infections and can be used to detect animals with clinical or subclinical infections. It is interesting to note that the evaluation of PMN phagocytic activity was higher in negative samples and

tended to increase during the lactation. The relation between infections and PMN phagocytic activity is comparable to what has been observed in cows where high PMN phagocytic activity was associated with a low level of infection. Our results are in agreement with Guidry and Paape (1976) who describe PMN activity as one of the ®rst mechanisms of mammary immune defenses that limit microorganism replication in the mammary gland of ewes.The evaluation of immune parameter threshold values and enzymatic kinetics applied to ewe milk will be the next step to evaluate the general level of ewe mammary gland defenses and it will help to identify the animals which are the most exposed to mammary infections. Acknowledgements We thank Dr. Silvia Bramani for critically reading the manuscript. References Bergonier, D., Lagriffoul, G., Berthelot, X., Barillet, F., 1994. Facteurs de la variation des comtages de cellules somatiques chez les ovins et les caprins aitieres. In: Proceedings of International Symposium Somatic Cells and Milk of the Small Ruminants Bella. Italy, 139 pp. Cuccuru, C., Zecconi, A., Moroni, P., Contini, A., 1996. Cellule Somatiche e Infezioni Mammarie Ovine. Obiettivi e Documenti veterinari, XVII, 73 pp. Cuccuru, C., Moroni, P., Zecconi, A., Casu, S., Caria, A., Contini, A., 1997. Milk differential cell counts in relation to total counts in Sardinia ewes. Small Rumin. Res. 25, 169±173. Dulin, A.M., Paape, M.J., Capuco, A.V., 1984. Activity of Nacetyl-b-D-glucosaminidase in milk: contribution by somatic cells. J. Dairy Sci. 67 (Suppl. 1), 253. Dulin, A.M., Paape, M.J., Nickerson, S.C., 1988. Comparison of phagocytosis and chemiluminescence by blood and mammary

P. Moroni, C. Cuccuru / Small Ruminant Research 41 (2001) 1±7 gland neutrophils from multiparous and nulliparous cows. Am. J. Vet. Res. 49, 172. FIL-IDF (Federation Internationale de Lactorie-International Dairy Federation), 1981. Laboratory methods for use in mastitis work. FIL-IDF Brussels. Bulletin, 132 pp. FIL-IDF (Federation Internationale de Lactorie-International Dairy Federation), 1995. Enumeration of somatic cells. FIL-IDF Brussels. Bulletin 148A, 195. Fruganti, G., Ranucci, S., Tesei, B., Valente, C., 1985. Safety evalutation of sheep udders during an entire lactation period. La Clin. Vet. 108, 286±296. Guidry, A.J., Paape, M.J., 1976. Effect of bovine neutrophils maturity on phagocytosis. Am. J. Vet. Res. 37, 703. Kaartinen, L., Mattila, T., Frost, A., Sandholm, M., 1990. Sequestration of N-acetyl-b-D-glucosaminidase in somatic cells during experimental bovine mastitis induced by endotoxin, staphylococcus aureus or streptococcus agalactiae. Res. Vet. Sci. 48, 306. Kirk, J.H., Glenn, J.S., Maas, J.P., 1996. Mastitis in a ¯ock of milking sheep. Small Rumin. Res. 22, 187±191. Kitchen, B.J., Middleton, G., Salmon, M., 1978. Bovine milk Nacetyl-b-D glucosaminidase and its signi®cance in the detection of abnormal udder secretions. J. Dairy Res. 45, 15.

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