This paper describes an extensive field investigation in the bulk-tank milk of sheep flocks, one of largest ever on worldwide basis. Dairy sheep farming is an important sector of the agricultural industry in Greece, with a significant annual milk production. In 2019, total deliveries of sheep milk to dairy factories were 643,027,000 liters [
15], accounting for approximately 20% of European and 15% of world sheep milk production [
16]; this milk is used mainly for cheese production [
16]. Sheep flocks from all regions of Greece were included into the study; that way, conditions prevailing throughout the country had been taken into account and factors of regional importance weighed less. In order to minimize possible bias, the study also used consistent methodologies and ensured that specific tasks were always performed by the same investigators.
Although sheep milk is of great importance for the Greek agricultural sector, no systematic countrywide investigations in the bulk-tank milk of sheep in Greece have been reported. Nationwide investigations of bulk-tank milk are important, because they allow for evaluation and monitoring of the quality of produced milk.
4.1. Somatic Cell Counts in Bulk-Tank Milk
In studies that appraised the relevant situation during the 1990s in the country, SCC over 1.0 × 10
6 cells mL
−1 in the bulk-tank milk from sheep were reported [
17,
18], values that are substantially higher than the ones found in the current investigation. Although it can be difficult to directly compare studies performed by differing methodological approaches, there is still merit in analyzing them, as they indicate the changes that have taken place within the last 20 to 30 years. This obvious reduction in SCC reflects the changes that have occurred in the Greek sheep industry during that period and the achievements in improving management of the flocks, benefiting from the general scientific progress in the field and the social changes in the country. The establishment of machine-milking in flocks has been an important factor that contributed in the improvement, as also corroborated in the present results (
Table 3). This was coupled to the extensive import of animals of Lacaune breed (in the current study, in 106 flocks), in which sustained efforts have been made to improve low SCC [
19]. The training of veterinarians active in the discipline has also improved and has led to increased implementation of udder health management practices, whilst improved training of the sheep farming community has also played a definite role (
Table 3).
It is interesting to note that higher SCC values have been reported in similar studies in other countries with prominent dairy sheep farming sectors. In a study in North Spain, the mean SCC in flocks was found to be 1.072 × 10
6 cells mL
−1 [
5], whilst in a study in Israel, the mean SCC in flocks was found to be 1.279 × 10
6 cells mL
−1 [
20].
4.2. Factors Potentially Affecting Somatic Cell Counts in Bulk-Tank Milk
The present study has assessed the possible effects of a wide range of factors on SCC. This was achieved with two types of analysis: the first to identify factors that may lead to higher SCC and the second to identify factors that may lead to SCC over 1.0 × 106 cells mL−1.
Although the current EU legislation does not mention a legal threshold for SCC in the milk of sheep, the threshold of 1.0 × 10
6 cells mL
−1 was considered and applied in this study, for two reasons: first, the work of Berthelot et al. [
21], who indicated that in milk samples from individual ewes the value of 1.0 × 10
6 cells mL
−1 confirms mastitis in the animal, and second, the use of this value by some Greek dairy factories to qualitatively classify milk produced in sheep flocks and regulate prices paid to farmers. This practice is in line with a similar approach applied in Spain, where dairy factories also classify milk according to SCC [
5]. Nevertheless, the bulk-milk threshold should be considered separately from the somatic cell counts in the milk of individual ewes, in which other values apply, for example Albenzio et al. [
22] have indicated that an impairment of mammary gland of individual animals can be observed in values as low as 0.3 × 10
6 cells mL
−1.
Mastitis is the most important and significant factor associated with high SCC and the cumulative evidence from the present study confirms that SCC in the bulk-tank are mostly dependent on the presence of mastitis in a flock, with some other factors found to having some significance. In flocks with a reported annual incidence risk of clinical mastitis > 0.5%, bulk-tank milk SCC were significantly higher and also this was the most significant factor identified in the multivariable analysis for SCC over 1.0 × 10
6 cells mL
−1 (
Table 3 and
Table 4).
Other factors that proved significant for SCC over 1.0 × 10
6 cells mL
−1 were the month into the lactation period and the age of the farmer. The former variable was found to be particularly influential at the start of the milking period (
Table 4). In previous studies [
23,
24], it has been found that the start of milking was a significant predisposing factor for the development of mastitis in dairy ewes; to a large extent, the present results confirm from a different viewpoint previous findings. Towards the end of a milking period, there was again an increase of SCC (
Table 3), but not to the height observed at the start of the milking period. That increase has been repeatedly reported to occur in SCC of the milk of individual animals [
25] and considered to occur even in the absence of infection [
26]; the present results indicate that this increase was not high enough to be important.
The age of the farmers can also be of importance for increased SCC, as many variables related to management can depend upon them. A farm’s productivity has been found to decrease progressively with farmers over 45 years of age [
27]. This can be explained when considering the findings of a New Zealand study, in which it was found that farmers older than 50 years were using fewer health management tools and using them less frequently than younger farmers, and this was the case even for procedures as basic as anti-clostridial vaccinations in sheep flocks [
28]. This finding could be of great importance in diseases, such as mastitis, that require complex health management. It is noted that education of farmers was also important; this can also be associated with age, as younger farmers would have received some vocational or higher training, hence having better skills in flock management.
A negative correlation of SCC with mean body condition scores of ewes in the flocks was also found, a result that at first may not seem indicative of any causation. Nevertheless, one should take into account that, in sheep, the primary factor influencing body condition score is nutrition [
29]. Suboptimal nutrition, which is reflected in a low body condition score, can be responsible for compromising sheep immunity through various pathways; these include the reduced formation of immunoglobulins, the inefficient cellular response, the lack of micronutrients necessary for the integrity of epithelia (e.g., zinc) or immune processes (e.g., selenium) [
30], all of which play a role in the efficient defenses of sheep against mastitis pathogens [
31]. Further support for this hypothesis comes from Barbagianni et al. [
32], where it was shown that suboptimal nutrition throughout the final stage of pregnancy predisposed ewes to mastitis during the subsequent post-partum period.
In other relevant studies, various factors were also reported to be influencing SCC in the bulk-tank milk. For example, in the study of Gonzalo et al. [
5], machine-milking, flock size, culling rate, administration of ‘dry-ewe’ treatment at the end of the lactation period, post-milking teat dipping were reported to influence SCC. In another study in Spain, only the season of sample collection (which, to some extent, is related to the month after start of the milking season) was found as a significant factor [
33], whilst in a third one machine-milking and administration of ‘dry-ewe’ treatment at the end of the lactation period were found as significant factors [
34]. In comparing those results to the present ones, we note that whilst some of the factors identified elsewhere were found to be of importance in our univariable models, they were not chosen by the multivariable analysis. These findings reflect the multifaceted and multifactorial nature of mastitis and the importance of the many predisposing factors [
31], many of which also have complex interactions between them.
4.3. Bacteriological Findings and Factors Potentially Affecting Total Bacterial Counts in Bulk-Tank Milk
There was a difference between the staphylococcal species identified in this study and the species generally confirmed as aetiological agents of subclinical mastitis [
35]. The present results indicate
S. simulans,
S. equorum and
S. haemolyticus, as the main species identified, whilst, in general,
S. epidermidis,
S. simulans and
S. chromogenes are the cnS species usually recovered from cases of subclinical mastitis. This study also recovered species that have not been considered as mastitis pathogens, e.g.,
S. lugdunensis. The above indicate that many of the staphylococci in the bulk-tank milk were not of sheep origin, but originated from other sources in the flock environment, possibly staff (
S. haemolyticus) or other animal species (
S. intermedius). This also indicates the possibility of contamination of the milk with bacteria of human origin, which then can act as potential human pathogens (e.g., with production of enterotoxins or transfer of antibiotic resistance genes). Albenzio et al. [
36] reported that the hands of milkers were the main sources of milk contamination with bacteria of non-animal source. Although in most cases pasteurization of milk would kill such bacteria, one should also take into account the cases of cheese production made of unpasteurized milk, mainly in small-scale local cheese types.
Among the staphylococcal isolates recovered, 71.5% were identified as biofilm-forming, which indicates the high proportion of such strains even among non-sheep sources. Increased adhesion properties can lead in colonization of the milk system (teatcups, milklines, etc.), thus providing increased risk for intramammary infection of ewes at the parlor, leading to staphylococcal mastitis [
14].
The difference in the sources of bacteria in milk is reflected in the difference of factors that can influence TBC to the respective factors for SCC in the bulk-tank milk. For example, water cleaning of the parlor is important (
Table 5), because it reduces bacterial load in parlor equipment (e.g., milklines) and thus contributes to reducing milk contamination. Moreover, even for factors that are of importance for both high SCC and high TBC, there may be differing reasons in their significance, for example, after the start of the milking period (more frequently occurring in autumn or winter), sheep spend a lot of time indoors and animal houses are crowded (which may also occur due to presence of lambs not yet sent for slaughter): this results in increased bacterial loads within the animal houses and facilitates milk contamination and high TBC. As the lactation period advances and animal houses become less crowded, bacterial loads decrease and this is reflected in lower TBC.
The current results indicate that in most cases raw milk from sheep flocks complied with the standards required in the legislation. The value of 1500 × 10
3 cfu mL
−1 is in the current EU legislation the acceptable upper limit of bacterial counts in raw milk from sheep [
6]. The findings indicate a significant reduction in total bacterial counts to those reported by Anyfantakis [
17] and Papadopoulos [
18], who indicated that TBC > 5000 × 10
3 cfu mL
−1 prevailed in raw milk from sheep farms in Greece during the 1990s. Again, the same reasons as for SCC would have contributed to this reduction. Sevi et al. [
37] have suggested that the threshold of 0.7 × 10
6 cells mL
−1 for bulk-tank milk from ewes, allows for low microbial burdens in the milk and the present findings are in line with that proposal.
There are some differences between the current results and those of Gonzalo et al. [
5]. In the current study, SCC were lower to those reported by Gonzalo et al. [
5], whilst TBC were higher (111 × 10
3 cfu mL
−1 [
5]). With regard to the factors potentially affecting TBC, there was a greater similarity between the two studies than for SCC, with administration of ”dry-ewe” treatment at the end of the lactation period and annual frequency of removal/clean-up of the straw bedding having been identified to be significant during the univariable analysis in both studies. The above further indicate that there is no complete association between SCC and TBC.
4.4. Associations with Milk Content and Milk Production
The adverse effects in the milk content found to be associated with high SCC are compatible with the effects of mastitis on milk composition of affected individual ewes [
38,
39]. The present study found that in cases of SCC over 1.0 × 10
6 cells mL
−1, the protein content in the bulk-tank milk was significantly reduced. This directly associates increased SCC in raw milk with reduced cheese production from such milk, given that protein content of milk is a primary determinant of cheese yield. In a similar approach, Sevi et al. [
37] have indicated that for SCC over 0.7 × 10
6 cells mL
−1 the renneting ability of milk would decrease.
When milk with high SCC is delivered, dairy factories can impose penalties in the price. The correlation of high SCC with increased water content in milk suggests that farmers might try to recuperate losses in the price of milk (and amount of money received) by increasing sales volumes through addition of water in the milk.
It was also found that in flocks with SCC in bulk-tank milk > 1.5 × 106 cells mL−1, there was a reported lower milk production per animal. This further increases the potential adverse financial effects of high SCC.