Mastitis is a major health problem in dairy sheep flocks worldwide. It is associated with reduced productivity, low weaning weights, lamb mortality and the culling of affected ewes. The lactation incidence of clinical mastitis in the ewe is generally lower than 5%, while the prevalence of subclinical mastitis is variable and ranges from 10 to 30% or more. Staphylococci are the main aetiological agents of intramammary infections (IMI), the more frequent isolates being S. aureus in clinical cases and coagulase-negative species (CNS) in subclinical IMI. Small ruminants mastitis is a chronic and contagious infectious: the primary sources are mammary and cutaneous carriages, and speading mainly occurs during milking. Somatic cell counts (SCC) represent a valuable tool for prevalence, assessment and screening. At individual level, the use of several successive SCC or the California Mastitis Test (CMT), and microbiological examinations allows the efficient detection of subclinical mastitis and is a good predictor of persistance. Progressive replacements combined with intramammary antibiotherapy at drying-off period are the main measures for the elimination of IMI. Prevention includes the control of environmental sources and bacteriological transmission during milking. The detection of the ovine genome regions involved in mastitis resistance will also include information on mastitis occurrence in addition to SCC. Appropriate genetic selection, together with the implementation of preventive measures, could reduce the negative consequences of those factors.
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Key words: ewe, mastitis, somatic cell count, epizootiology
Mastitis can be defined as an inflammation of the mammary gland or udder of the ewe due to various causative factors. It is characterized by increased SCC and abnormal findings in mammary tissue. In the ewe, the main causes of intramammary infection (IMI) are bacteria, mycoplasma spp. or lentiviruses. In most cases, mastitis is of bacterial origin. Mammary pathogens are classified as contagious, environmental and other coagulase-negative staphylococci (CNS). Besides the causative microbiological factor, important role in causing mastitis have predisposing factors such as environmental, genetics, anatomic and nutritional. From a clinical perspective, mastitis takes several forms: clinical mastitis can be identified by abnormalities in the udder such as swelling, heat, redness, hardness or pain. Other indications may be abnormalities in milk such as a watery appearance, flakes, clots, or pus. Subclinical mastitis is characterized by no clinical signs and quantitative and qualitative changes in milk composition. Mastitis in ewes occurs in all countries of the world where sheep are kept, but most attention has been paid to the disease in those countries in which there are substantial milking flocks e.g. the Mediterranean region. It is important for mainly economic reasons and has negative effects on both milk quality and animal welfare. Most of the information that is available relates to dairy cattles. Validation of specific control programs against mastitis is based on knowledge relating to dairy cattles IMI. Important differences exist between two species. Current breeding programs in dairy ewes is based on selection for high milk yield. This fact, combined to intensive management schemes increased the incidence risk of mastitis and effected negatively on milk production. The approach to mastitis control should be carefully made with a specific point of view, and not by generalizing results obtained from research on mastitis in dairy cows. The purpose of this paper is to review recent research into mastitis in dairy ewes, the methods of diagnosis; current control measures and that molecular genetics technology is an important tool for assessing susceptibility to mastitis.
Traditionally, Staphylococcus aureus has been regarded as the principal cause of mastitis in dairy ewes. This bacterium is responsible for both clinical and subclinical mastitis. The results of studies of clinical mastitis in dairy ewes show the high prevalence of Staphylococcus aureus (Albenzio et al., 2002; Ariznabarreta et al., 2002; Contreras et al., 1999; Kanellos and Burriel, 2002; Pengov, 2001; Suarez et al., 2002). By producing toxins, such as enterotoxins and leukotoxins, Staphylococcus aureus contributes to the pathogenesis of mastitis (Rainard et al., 2003).
Coagulase-negative staphylococci (CNS) are the most prevalent pathogens causing subclinical mastitis in dairy ewes. Some strains have even been isolated from clinical mastitis cases (Fthenakis and Jones, 1990). They can also produce persistent subclinical mastitis, significantly increase SCC, and cause clinical mastitis, although they are less pathogenic than S.aureus (Fthenakis, 1994; Deinhofer and Pernthaner, 1995; Contreras et al., 1997; Ariznabarreta et al., 2002). Among the CNS, S. epidermidis, S.xylosus, S.chromogenes and S. simulans are the more frequently isolated in ewes (Gonzalo et al., 2002; Contreras et al., 2003; Cuccuru et al., 2011). Other species that have been isolated are S. haemolyticus, S. fleuretti and S. sciuri. In goats, S. caprae is one of the most prevalent species. Because the pathogenicity of the different CNS species varies widely, some authors suggested segregation of CNS in two groups: novobiocin sensitive (NSCNS), which associated with mild changes and novobiocin resistant (NRCNS), leading to numerous modifications of the quality and quantity of milk. A recent study of Gonzalo et al. (2000) showed that NSCNS were associated with high SCC compared with NRCNS.
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2.2 Mannheimia haemolytica
The most causative agent of mastitis in ewes suckling lambs is Mannheimia haemolytica (Menzies and Ramanoon, 2001; Bergonier et al., 2003). The incidence of the organism is smaller when ewes are milked (Bergonier et al., 2003). Scott and Jones (1998) reported the isolation of Mannheimia haemolytica from the teat skin of suckling lambs, but not from that of ewes before lambing or after weaning. The bacteria entries from the teat orifice and ascends to the mammary gland through the teat (Bergonier and Berthelot, 2003
2.3 Mycoplasma spp.
Contagious agalactia is characterized by symptoms other than mastitis; some authors fail to consider Mycoplasma spp. as etiology of sheep IMI. Mycoplasma agalactiae is the major etiological agent of this disease. In recent studies some other species such as M. putrefaciens and M.mycoides have been isolated (Bergonier et al., 1996). The disease is indicated by pyrexia, keratoconjunctivitis, arthritis (swollen joints). Infection is followed by bilateral mastitis and changes on both milk quality and quantity (yellow, granular and with clots milk) (Corrales et al., 2004). In chronic cases, mammary gland becomes atrophied. The intense effects of Mycoplasma spp. in reducing milk production and increasing the SCC, means that contagious agalactia should be considered as one of the most important causes of mastitis in endemic areas, where clinical cases are frequent.
2.4 Other bacteria and fungi
In a decreasing order of frequency, isolates belong to Streptococci, Corynebacteria, Pseudomonas spp. In addition, severe cases of mastitis have been attributed to the pathogens Aspergillus fumigates, Serratia marcescens, P.aeruginosa or Burkholdelia cepacia (Las Heras et al., 1999; Berriatua et al., 2001; Bergonier and Berthelot, 2003; Contreras et al., 2003; Gonzalo et al., 2004b). Winter et al., (2004) reported one case of clinical mastitis in a ewe caused by L. monocytogenes. The most important results of this study were a highly increased SCC and persistent shedding of bacteria through milk. Some other studies mentioned the importance of Nocardia spp. isolation, due to their potential for causing disease in humans, and because Nocardia farcinica causes mastitis in goats (Berriatua et al., 2001; Maldonado et al., 2004).
Lentiviruses are also known to infect small ruminants, but they are not usually considered as classic intramammary infections, because they rarely produce clinical symptoms or elevated SCC (Turin et al., 2005). Pekelder et al., (1994) noticed that in affected flocks, Maedi causes slowly progressive, indurative mastitis. There are no changes in milk quality, except fat which usually decreases. The mastitic form of the disease coexists with the other forms (nervous, articular and respiratory).
3. Descriptive and analytical epizootiology
3.1 Incidence, prevalence and persistence
The incidence of clinical mastitis in dairy ewes is generally lower than 5%, but this incidence can increase sporadically. However, some authors reported a few outbreaks where the incidence ranged from 30% to 50%, causing mortality or culling of the ewes (Lafi et al., 1998; Calavas et al., 1998; Haenlein, 2002). Data from several sources have identified the increased incidence of clinical mastitis in early lactation followed by stable incidence until weaning (Bor et al., 1989; Mork et al., 2007; Arsenault et al., 2008). A recent study by Gougoulis et al. (2008), tried to explain this pattern. The results showed that bacteria, present in lamb's mouths, could be mechanically transmitted to ewes. Incidence of subclinical mastitis is also high at the beginning of lactation (Kirk and Glenn, 1996; Leitner et al., 2001; Bergonier et al., 2003).
The prevalence of mastitis is estimated by means of bacteriological analysis of half-udder or udder milk samples, individual somatic cell counts (iSCC) or the CMT. The reported prevalence ranges from 5 to 30% per lactation (Lagriffoul et al. 1999; Bergonier and Berthelot, 2003; Berthelot et al., 2006). Bulk milk somatic cell counts (bSCC) can also be used to assess the prevalence of mastitis at the flock level. This method considers as the only easy and cheap way to estimate the whole flock/herd mammary infectious status (Lagriffoul et al. 1999; Bergonier et al., 2003). On the other hand, in goats the relationship between bSCC and the prevalence of mastitis is rather difficult to determine, due to effect of lentiviruses and non infectious factors on SCC. Fahr et al., (1999) reported the influence of the kidding season on the bSCC values.
The persistence of clinical mastitis during lactation depends on the production type and on the technical level. Kirk and Glenn, (1996) reported that mastitic ewes are rarely culled. The main reason is that cases of acute mastitis become chronic leading to persistence of several months, or even for a whole lactation. In literature, there are a few data. Both in ewes and goats, mammary pathology is the first cause of culling for sanitary reasons; it is more frequent during the first 2-3 months of lactation. The persistence of subclinical IMI during the dry period is important to consider regarding the treatment strategies. Esnal et al., (1994) reported an overall self-cure rate of 20 to 67% in the ewe. Despite the high incidence of CNS linked to IMI in sheep, the pathogenic mechanisms that underlie the subclinical infections remain largely unknown. Using S. epidermidis to induce IMI in ewes, Winter and Colditz (2002) reported that lactating udders are capable of a prominent local inflammatory response.
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Silanikove et al., 2005 have shown that sheep are more susceptible than goats to milk yield losses due to subclinical mastitis. In addition, when comparing goat and sheep IMI, it seems that the sheep mammary gland is more affected by CNS (Leitner et al., 2004a, b). The hypothesis of different defense mechanisms for the goat udder than for the bovine udder has been considered (Menzies and Ramanoon, 2001). Some authors suggest that in goats compared to dairy cattle, the low incidence of clinical IMI might be related to the physiologically high SCC and PMNL percentage. This hypothesis is weakened by the comparison with the ewes situation: the same low clinical IMI incidence and, on the contrary to goats, the same range of somatic cell counts and percentages are recorded in cattle.
In order to minimize hazards that could be caused by bacteria, milk is generally heat treated. However, in regions where cheese is made from raw milk, controlling mastitis becomes a priority. Staphylococcus aureus produces thermostable toxins which have the ability to survive even in pasteurized milk. In addition to enterotoxins produced by S. aureus, there is also a wide pattern of virulence factors such as the leukotoxins. These leukotoxins can selectively destroy (PMN) and monocytes. Rainard et al., (2003) investigated the leukotoxic actions of S. aureus strains isolated from cows, sheep and goats with mastitis. The results showed that strains isolated from small ruminants were more leukotoxic towards bovine PMN than those from cows.
3.2 Effects of lactation stage and number
3.2.1 Lactation stage
Higher rates of clinical IMI incidence are observed at the beginning of lactation, during suckling-milking period and particularly at the beginning of exclusive milking. The main reasons are machine milking and infections due to S. aureus (Bergonier et al., 2003). A high incidence of subclinical mastitis was reported at the beginning of lactation (Kirk and Glenn, 1996; Leitner et al., 2001). During drying-off period, low rates of IMI incidence are rarely observed. Nevertheless, some authors mentioned that in sporadic and rare cases due to mycotic agents or P.aeruginosa, a higher incidence is recorded (Las Heras et al., 2000, Leitner et al., 2001).
3.2.2. Lactation number
An increased prevalence related to parity has been reported in ewe. Leitner et al., (2001) mentioned a tendency for prevalence to increase as the parity increased. Similar results were reported by Sanchez et al. (1999) for goats. On the other hand, Sevi et al. (1999) reported that mastitis infection set in progressively earlier as parity decreased.
3.3 Susceptibility factors
Staphylococci are normally carried by teat skin. However, their main sources are infected udders, injured teats and viral lesions. Enterobacteria and Enterococci are found particularly in the litter, and Pseudomonas spp. especially in water. Ziluga et al., (1998) reported the isolation of Mannheimia haemolytica both in the respiratory tract (nasopharynx and tonsils), as much in the environment. Other primary sources are environmental: A. pyogenes and A. fumigatus are isolated from mouldy forage, wet bedding, litter, and air. S. uberis and S. suis are isolated from multiple sources: infected animals, litter, and the environment. Infection from a primary source is usually temporarily followed by secondary complications. The milking equipment and milker's hands during hand milking are playing a prospective role which should be pointed out (Ziluga et al., 1998; Burriel, 1998).
3.3.2 Associated factors
The inability of early mastitis detection is the main factor that contributes to bacterial conservation in infected udders. As post-milking teat antisepsis is carried out only occasionally, infected teat lesions also represent dangerous sources. Poor maintenance of milking equipment contributes to the persistence of bacteria. In a review, Bergonier et al., (1997) mentioned the persistence of P. aeruginosa in the milking equipment even when the cleaning and disinfecting procedure has been correctly applied. Finally, inappropriate design and use of the pen (area per ewe, ventilation etc.), can lead to bacterial infection.
3.3.3. Udder morphology and health
The relations between udder morphology, especially teat placement and orientation, milkability and SCC have been studied in certain breeds (Marie et al., 1999). According to Casu et al., (2010), animals with deep and pendulous udder and with high implanted teats are more susceptible to udder inflammation or mastitis. Gelasakis et al., 2011 reported that in Chios breed ewes, the combination of small size and horizontal position of teats together with the inappropriate udder conformation can predispose to mastitis. Recently, Kiossis et al. (2009) indicated that the most common finding at the teat cistern, using teat endoscopy, was the nodular proliferation of the mucosa. Histopathological examination revealed a chronic inflammatory process. Similar researches have been performed in sheep, showing that these proliferations are associated with intramammary infections (Mavrogianni et al., 2005; Fragkou et al., 2007).
3.3.4 Environmental factors
Milking is the main factor for microorganisms spreading. During lactation, the main factors for receptivity are the milking equipment, the milking routine and suckling. Thus, liners can induce repeated traumas, microhaemorrhages, congestions, if the vacuum level or the pulsation characteristics are inadequate. Bacteria can also transport passively by liners. Since the automatic cluster is developing nowadays, cluster removal without previous vacuum cutting off may also induce impact. To this direction, over milking can induce teat lesions. Transmission is also possible by «milk-robber» lambs and may be important for staphylococci, Pasteurellaceae, parapox virus (contagious ecthyma), etc. During the suckling period, orphan lambs and lambs underfed by mastitic ewes, may injure the teats of the ewes they try to suckle. Drying-off is not a period of risk for new intramammary infections in the ewe, but teat duct integrity can be affected by traumatic canula insertion, leading to a higher receptivity. Marco Melero (1994), using CMT to detect subclinical mastitis, observed a higher prevalence in hand-milked flocks (32.3%) than in machine-milked flocks (28.8%) in relation to the annual mean bSCC (847 and 664,000 cells per ml, respectively). Therefore, the incidence of acute mastitis and mammary abnormalities was higher in machine-milked flocks. Persistence is conditioned by the quality of mastitis detection and the application of control measures (treatment, culling) by the farmer.
3.3.5 Genetics of mastitis
There are several estimates of the heritabilities (h2) of SCC and other milk traits in sheep. An estimate of its heritability indicates the extent to which a trait is under genetic control, on a scale from 0 to 1 (with 0.6 being a high heritability and 0.05 being low). The h2 values for SCC are relatively low, with an average of 0.14, ranging from 0.04 to 0.24, depending on the breed and stage of lactation when they were estimated. All the estimates are from dairy sheep breeds. Its genetic correlations with other production traits show that there is no consensus among the estimates for the genetic relationships between milk yield and SCC, unlike those for dairy cattle. It is possible that the genetic improvement in dairy sheep breeds has not been as great as that in dairy cattle and that any susceptibility among high-producing sheep to mastitis is not yet apparent. It is also possible either that there is no antagonism between selections for increased milk production and reductions in SCC in sheep, or that the genetic susceptibility to mastitis of high-yielding sheep has been masked by the management strategies employed to cope.
3.3.6 Nutritional factors
In dairy ewes, the lack of vitamin E and selenium during the dry period may predispose to mastitis. Ronchi et al., (1996) demonstrated that the parenteral administration of them can reduce SCC and increase the percentages of milk neutrophils during the subsequent lactation.
4.1 Clinical symptoms
Clinical mastitis is generally easily diagnosed because of the characteristic clinical symptoms in the udder, the reduction in milk production and the changes in the composition and appearance of the milk. Initially, the mammary gland is controlled with an overview. The presence of skin and teat lesions and the temperature of the ewe are recorded. The existence of local signs and udder abnormalities (udder asymmetry, sclerosis, abscess, etc.) should be detected by inspection and palpation. Functional signs should be observed, at the beginning of milking, by foremilk inspection in a bottom container in order to estimate the composition, the content and the color of exudates. Milk samples are collected for bacteriological examination. The detection of mastitis, should lead to the identification of infected udders (which are sources of bacteria) in order to decide their treatment or culling. Some authors revealed that such a diagnosis should be performed systematically at least at the beginning and end of lactation (Kirk and Glenn, 1996; Esnal et al., 1999).
4.2 Bacteriological analysis
Bacterial culture is necessary for the identification of mastitis' causative agent in dairy ewes. Milk samples are microbiologically examined in order to ensure that the potential pathogens came from the inside of the mammary gland and not from dust or faecal particles on the udder surface. Samples should be kept refrigerated from the moment of collection until bacteriological examination. Most of the bacteria pathogens causing mastitis grow on ox or sheep blood agar. If there is suspicion of the involvement of Mycoplasmma spp. and Listeria spp. the use of specific substrate for cultivation and isolation is recommended. For economic and practical reasons, usually only one milk sample is used for the diagnosis of IMI.
Milk SCC is a marker of udder inflammation and measures the different cell types present in milk. Previous studies have reported that immediately after the entry of pathogens in mammary glands, leucocytes and epitheliac cells initiate the inflammatory response so that to eliminate the invading bacteria (Paape et al., 2002, 2003; Rainard and Riollet, 2003). A considerable amount of literature has been published on milk cell population. These studies have shown that in ewes, macrophages are the predominant cell type (45-88%). Polymorphonuclear neutrophil leukocytes (PMNL) constitute 6-20 % and lymphocytes 11-20%. Epithelial cells are present in small numbers (1-2%) (Menzies and Ramanoon, 2001; Paape et al., 2001; Haenlein, 2002; Bergonier et al., 2003; Blagitz et al., 2008). Due to their apocrine milk secretion, cytoplasmic particles are components of dairy ewe's milk. They are similar in size to milk somatic cells. Their average concentration in milk is 150-103/mL for goats and 15-103/mL for ewes (Paape et al., 2001). A number of studies have reported about variation of milk cell population during lactation. In 1996, Morgante et al. showed an increase in macrophages and eosinophil towards the end of lactation. One year later, Cuccuru et al., (1997) demonstrated an increase of macrophages and PMNL during lactation, whereas lymphocytes and epithelial cells decreased. In bacteriological negative ewe's milk, epithelial cells seem to be less than 2-3% of somatic cells, polymorphonuclear neutrophil leukocytes constitute 10- 35%, macrophages 45-85% and lymphocytes 10-17%.
An instantaneous relationship between the isolation of bacterial species and SCC has been reported in various studies, concluding that the distinction between major and minor pathogens, formerly used in the cow, is not relevant to ovine mastitis. Berthelot et al. (2006) suggest the use of individual SCC (iSCC) in combination with microbiological examination as a useful diagnostic tool for subclinical mastitis. The kind of screening, using iSCC methodology, can be adapted to operational purposes (detection for culling, drying-off treatment, etc.) and sometimes to IMI prevalence assessment (Mc Dougall and Voermans, 2002). Usually, in field conditions, a few iSCC values are available per lactation. The main advantage of such an approach is the individual treatment decision. Adversely, bulk SCC (bSCC) cannot give a clear picture of udder health status, especially in cases that negative microbiological examination is recorded. According to literature, there are two types of approach for the practical use of SCC. The first proposes an instant threshold value which distinguishes between «healthy» and «infected» udders. The second proposes the use of several SCC during lactation and defines three categories («healthy», «doubtful» and «infected»).
In recent studies, the majority of proposed threshold values allowing the discrimination between healthy and infected udders or halves are lower than 500 - 103 cells/ml (González-Rodriguez et al., 1995; McDougall et al., 2001; Paape et al., 2001; Pengov, 2001; Gonzalo et al., 2002; Bergonier and Berthelot, 2003; Berthelot et al., 2006; Blagitz et al., 2008). At the past, some authors have suggested different values, ranking up to 1500 - 103 cells/ml (Fthenakis et al., 1991; Mavrogenis et al., 1995).
Studies proposing a decision rule including a class of doubtful udders originate from a comparison of SCC performed every month during lactation in contribution of monthly bacteriological analyses of milk samples from udder-halves. An udder is considered healthy if every SCC is below 500 - 103 cells/ml, infected when at least two SCC are over 1000 - 103 cells/ml and doubtful in other cases (Bergonier et al., 1999). These efficiency rates and thresholds are similar to those for dairy cow (Serieys, 1985). Finally, a recent study by Spanu et al., (2011) revealed the importance of the control of SCC in the ewes. According to the results, milk samples obtained from ewes with three or more monthly SCC ≥ 400 - 103 cells/ ml during previous lactation periods were 5.6-7.5 times more likely to be microbiologically positive for mastitis pathogens compared with milk samples obtained from ewes with MSCCs below this threshold. That was similar to the results of Riggio et al. (2009), who mentioned that increased SCC lead to greater culling possibility.
Today, most dairy laboratories use SCC methods (fluor-optoelectronic counters) that are adequate for dairy ewes milk. At present, the direct microscopic SCC method using methylene blue staining is the reference method recommended by the IDF (1995). On the other hand, according to Paape et al., (2001), this method can overestimate the SCC of goat milk due to high concentrations of cytoplasmic particles. However, given that the SCC is an indicator of milk quality and that bonus/penalty schemes for the dairyman are based on the bulk tank SCC, it is important that the SCC is as accurate as possible. Some countries, like U.S.A. had more specific reference methods in order to obtain accurate SCCs, as it is the pyronin-Y Methyl green stain (Haenlein and Hinckley, 1995; Paape et al., 2001; Haenlein, 2002; Contreras et al., 2007). Previous studies, have descriped the significant correlations between the Fossomatic method and DNA specific strains (Menzies and Ramanoon, 2001; Gonzalo et al., 1993; Gonzalo et al., 2004); two methodologies with comparable results (Haenlein, 2002). Similarly, the calibration of somatic cell counters for use in small ruminant's with cow milk standards has been discussed by Zeng et al. (1999), who demonstrated an overestimation of goat SCC compared to somatic cell counters calibrated with cow milk standards. Sanchez et al., (2005) demonstrated that bromopol is a suitable preservative for goat milk samples refrigerated for as long as 25 days or frozen for 25-105 days.
There are several non-infection factor that influence SCC such as parity, lactation stage (Paape et al., 2001; Menzies and Ramanoon, 2001; Haenlein, 2002; Bergonier et al., 2003; Luengo et al., 2004), breed (Leitner et al., 2001; Paape et al., 2007), milk production, litter size (Paape et al., 2001; McDougall et al., 2001), milking frequency (Nudda et al., 2002; Paape et al., 2007), machine or hand milking (Sinapis, 2007), estrus (Haenlein, 2002; Paape et al., 2007) and seasonality (McDougall et al., 2001; Paape et al., 2001). The mammary gland of the goat and ewe is also influenced by lentiviral infections, but an additional difference between the two species is reported. In 1999, Lergottaglie et al. reported that there are no evidences about the influence of Maedi-Visna infection on SCC. On the other hand, there is more information about the relationship between CAEV and SCC. A considerable amount of literature has been published about the relationship between serological status and bacterial IMI in herds with a high prevalence of CAEV and IMI (Hincley, 1990; Ryan et al., 1993).
4.4 California Mastitis Test (CMT)
The California Mastitis Test (CMT) indirectly detect the presence of increased numbers of leucocytes in milk. It allows a semi-quantitative evaluation of SCC after the addition of a reagent to the milk sample. There is a large volume of published studies describing the evaluation of sensitivity and specificity of CMT (Hueston et al., 1986; McDougall et al., 2001; Clements et al., 2003). The concordance between CMT and bacteriology is close to 80% (Fthenakis, 1995; Gonzalez-Rodriguez et al., 1996). In some studies, the results suggest that the negative predictive value of CMT is greater than the positive predictive value (Marco Melero, 1994). In conclusion, in the ewe, the CMT is a very reliable, useful, easy to perform and inexpensive indirect method to measure SCC. It must be carried out before milking to take into account the SCC variations associated with the milk fractions (Peris et al., 1991).
5.1 General principles
The combination of speed and efficiency is the only rule for mastitis treatment. There are two operating axes: a) the immediate initiation of therapy and b) intramammary antibiotherapy combined with subcutaneous or intravenous administration in cases where generalized signs coexist. The first purpose is to save the animal's life and, possibly, to save the diseased halves. A failure to treat in the early stage of the disease increases the dispersion of infectious agents in breeding. Even so, in some cases the result is often disappointing and may not result in recovery of the affected gland. Most of the information that is available on literature relates to clinical observations adapted from results obtained in dairy cattles; control-case studies in ewes are rare. In small ruminant field conditions, clinical and subclinical IMI treatments must be distinguished, the average treatment cost per animal being high in comparison with the culling value and the expected recovery. In some areas, antibiotherapy is more used at drying-off period, particularly for milk cellular quality control (Poutrel et al., 1996).
5.2 Intramammary antibiotherapy
Treatment should always include the intramammary administration of antimicrobial agents. There are a large number of formulations which contain a wide spectrum or combination of narrow range antibiotics. Given that very few of them have special approval for small ruminants, products with approval for cattles can be used. No results from a controlled trial are available on the efficacy (i.e. bacteriological and clinical cure) of intramammary antibiotherapy. Under field conditions, this kind of treatment is not used frequently for economic and practical reasons. Various studies have been published on antibiotic pharmacokinetics in the ewe; allow proposing therapeutically designs (Ziv and Soback, 1989). Nowadays, beta-lactamines and macrolides are widely used under field conditions. The economic interest of these treatments and also of new antimicrobial drugs (fluoroquinolones) use remains to evaluated. Each time, the economic costs of such treatments will be considered, with only a high genetic value justifying costly and/or time-consuming therapeutic measures.
5.3 Systemic antibiotherapy and complementary treatment
The systemic administration of antimicrobial agents is required a) in cases of peracute mastitis, whenever there is possibility of bacteremia, b) in cases of subacute mastitis and c) to treat abscess in mammary gland.
Bergonier and Berthelot, (2003) mentioned that complementary treatment may be implemented by the parenteral administration of anti-inflammatory drugs, frequent milk-out of the affected gland and/or oxytocin (3 to 5 IU by the subcutaneous route). The purpose of the treatment is to reduce the severity of clinical symptoms and improve ewe's welfare.
5.4 Withdrawal period and residues
During lactation, the infusion of a cattle-labeled formulation containing amoxicillin, clavulanic acid and prednisolone led to the detection of residues up to 136 h in the ewe and 112 h in the goat after the last infusion (Buswell et al., 1989). These results justify the 7-days withdrawal period prescribed by the European regulation after an out-of-label intramammary treatment in lactating small ruminants. According to Chaffer et al. (2003), after drying-off treatments of dairy ewes with a cattle-labeled formulation containing penicillin, nafcillin and dihydrostreptomycin, residues were detected at lambing in four (respectively five) ewes out of 190 (respectively 25) and no more after three (respectively 5) days. Considering the dry period durations, it can be assumed that the risk of residues in milk is virtually null at lambing and at the first milk delivery after a one to two-month nursing period.
6. Control and prevention
6.1 Breeding management
The main measures for the elimination of IMI are progressive replacements of the affected ewes combined with intramammary antibiotherapy at drying off period (Saratsis et al., 1998). Ewes affected by clinical mastitis must be segregated from the flock until culling. Ewes which still presenting chronic signs at the next lambing should be culled before the beginning of milking.
In order to avoid teat injuries (viral or traumatic) and secondary bacterial contamination (by teat antisepsis) the control of cutaneous sources is required. In addition, flocks with incidence rates of contagious ecthyma or staphylococcal dermatitis must provide parenteral antibiotherapy to control milk contamination.
Preventive husbandry and appropriate managemental procedures will improve the health status of the herd. In a recent study, Gonzalo et al., (2005) mentioned the improvement of the udder health of dairy sheep herds by optimizing milking machine standards and parlor systems. Most of the milking routines relate to dairy cattles, but they are implemented for small ruminants especially when the herd shows a high incidence of IMI. To control CNS-induced IMI, all milking routines should be revised and milking equipment must be periodically checked to ensure correct milking variables such as vacuum level, pulsation rate and ratio, vacuum reserve per milking unit, etc. The following values may be proposed or are recommended: 36 kPa vacuum levels, 180 pulsations per min and 50% pulsator ratio (Gonzalo and Marco, 1999). On the contrary, milk retention must be reduced through adaptation of the equipment to animal yield, teat size and flock size (Fernandez et al., 1997): vacuum pump capacity, vacuum reserve, claws volume and position with regards to the liners. Automatic cluster removal systems, when they exist, must be carefully set. Within the conditions of strict hygiene, adequate quality control of the water used to clean the milking equipment is needed to avoid infection outbreaks. Las Heras et al., (1999) reported a case study for infection by P. aeruginosa. Prevention of new intramammary infection can be achieved by using post-milking teat dipping, mainly in highly infected herds, and it has been revealed as a very effective method (Paape et al., 2001; Bergonier and Berthelot, 2003; Contreras et al., 2003). However, Tzora and Fthenakis, 1999 reported mastitis cases by S. marcescens when using a quaternary ammonium based teat dip. Thus, the quality control of the teat dip disinfectant is very important, because some sporadic outbreaks have been related to an inadequate disinfectant acting as an infection source. Conventional teat dipping products are either iodine or chlorine based. In one study, a dodecyl benzene sulfonic acid spray failed to maintain and/or restore the udder health of a sheep herd subclinically infected by CNS (Klinglmair et al., 2005). In sheep with IMI due to M.haemolytica, suckling lambs are the main source of infection as they spread the infection to their mothers. Weaning leads to a drop in the incidence of M.haemolytica mastitis. The use of pasteurized colostrums is on the increase in modern dairies for production and health reasons, such as combating lentivirus infection (Contreras et al., 2004).
Although that they are no evidences that vaccines available on the market for small ruminants are effective, they are widely used when there is a high incidence of clinical gangrenous mastitis. The effectiveness of vaccination programs against mastitis caused by S. aureus has been reported for sheep (Amorena et al., 1994, Tollersrud et al., 2002). The efficacy of a vaccine in preventing mastitis by S. aureus and S. simulans was assessed in field conditions (Marco Melero, 1994). The results indicated a reduced prevalence of clinical mastitis but not of subclinical infections. According to studies there are any reliable reports about the effectiveness of these vaccines and their inability to prevent new infections. Thus, it has been suggested that should be used in dairy herds with a high prevalence of S. aureus IMI in order to reduce clinical symptoms. A staphylococcal vaccine conceived and experimentally assessed in Spain (Amorena et al., 1994), was tested in a field trial in the Latxa breed. The results showed that there was not a significant difference in the prevalence of intramammary subclinical infections between vaccinated and control groups during the entire lactation, but the frequency of clinical mastitis was reduced (Marco Melero, 1994). There are any proven means about the efficiency of auto vaccines in controlled trials. Vaccination does not appear to be a decisive tool for the prevention of intramammary infections in the ewe.
6.3 Dietary measurements
Susceptibility testing of the udders should be based on dietary recommendations: limiting sudden transitions or imbalance. Giadinis et al. (2011) reported the likelihood of ovine mastitis in cases of Se deficiency. Se status of ewes may possibly indicate animals at risk to develop clinical mastitis; this may be the result of impaired cellular defense functions. Additional, reduced vitamin A serum concentration may also contribute to development of clinical mastitis. Vitamin A deficiency can lead to impaired epithelia integrity and, thus, to increase bacterial entry into the mammary gland; increased risk of developing mastitis.
6.4 Intramammary treatment at drying-off
Drying-off treatment considered to significantly reduce the incidence of IMI in dairy ewes and goats (McDougal and Anniss, 2005; Gonzalo et al., 2004). Poutrel et al., (1997) pointed out, that generalized intramammary antibiotic should be applied in high prevalence conditions. In addition, some authors noted that selective dry-off antibiotic therapy seems to be preferable than generalized, based on certain findings such as the spontaneous cure rate at parturition, which can be especially high for small ruminants, about 20-60% (Paape et al., 2001; Contreras et al., 2003; Bergonier and Berthelot, 2003). The aims of drying-off treatments in the ewe are bacteriological cure of infected udder-halves and prevention of new infections. Some authors indicated that the overall cure rate ranges from 65 to 95.8% in the ewe (Ahmad et al., 1992; Chaffer et al., 2003). The effectiveness is better for CNS than for S. aureus infections. The preventive interest remains to be discussed, particularly in ewes, considering the length of the dry period. Different treatment strategies are performed regarding the target for antibiotherapy. Additionally to the dry period length three peculiarities should be taken into account: the large herd size, the production cycle synchronization inducing a collective drying-off, and from a pathological point of view, the low drying-off and peri partum IMI incidences. Udder examination and iSCC or CMT are helpful to select the ewes requiring an antibiotic treatment. Ziv and Soback, (1989) reported that the implementation of intramuscular antibiotherapy (drying-off) in large flocks, would be an interesting route because it allows an easier implementation and a reduction of the «iatrogenic» risk of udder contamination.
The presence of different CNS species affects the choice of certain practices for controlling mastitis, such as the protocol and type of disinfectant used for teat dipping or dry-off treatments.
6.5 Mastitis control programs
Numerous studies have attempted to explain the necessity of adequate programs to control mastitis in order to improve udder health status and restrict bulk tank SCC (Contreras et al., 2003; Gonzalo et al., 2005). Nevertheless, Kiossis et al. (2007) reported a program which resulted in limitation of subclinical mastitis during lactation and a better health status for udders entering the dry period. Additionally, more and better quality milk was produced. In a recent study, Gonzalo et al., (2004b) mentioned that some cooperatives or association of small ruminant dairy farmers have started to develop programs of farm audits and the use of farm guidelines for mastitis control. The aim of this act is to help dairy technicians and dairymen optimize the mammary health of the flocks and to improve milk quality. Within the next few years a priority will be the defining of specific standards of milk hygiene and quality of small ruminant milk, and implementation of good management guidelines for these species. In order to ensure adequate and hygienic administration, of the antibiotic treatment, the presence of the veterinarian is required. Overuse of antibiotics increases the risk of antibiotic resistance and has become a public health problem. Mavrogianni et al., (2004) mentioned that the use of antibiotics registered for cows in small ruminants or even the use of drugs registered for ewes in goats, is possible to cause risks because of the unknown safety and efficacy of them. The absence of antibiotic residues in milk from cows and other species is mandatory in the European Union. Yamaki et al., (2004) demonstrated that positive results are higher in milk from small ruminants than in cow's milk.
6.6 Genetic resistance
Barillet et al. (2001) carried out a genetic analysis for mastitis resistance in the french Lacaune breed. The goal of this study was to define a breeding strategy for udder health in dairy sheep. The results showed a low frequency of clinical mastitis. Hence, selection for mastitis resistance in dairy sheep could be limited, at the moment, against subclinical mastitis. Such selection may be achieved using the indirect SCC trait. In another studies, results revealed a strong evolution of the genetic relationship between SCC and milk yield during the first lactation (Rupp et al., 2006; Barillet et al., 2007). Further analyses are necessary to validate these results. Confirmation of this assumption would allow designing a strategy based on a test-day approach instead of a lactation approach.
Breeding for resistance to mastitis in dairy cattle has been the subject of research over at least the past 20 years. Selective breeding for mastitis resistance is a sustainable method for its control and requires a suitable selection trait or molecular genetic marker, with sufficient additive genetic variance for this trait. In addition, effective breeding requires knowledge of the genetic relationship of the trait with other traits of economic importance. Because the SCC is genetically highly correlated with mastitis, selection for reduced SCC will also reduce the incidence of mastitis. However, there is considerable evidence that there are unfavorable genetic correlations between resistance to mastitis and milk production traits. According to Mark and et al. (2002), 12 countries have included mastitis in breeding programs alongside conventional traits to try to reduce the increases in genetic susceptibility to mastitis induced by selection for increased milk yield. Veerkamp et al. (1998) estimated that selection to decrease mastitis or limit the rate of increase of mastitis, with a cumulative impact of 1 per cent per year and a national penetration of 50 per cent, would result in a national benefit of € 0.8 million per year, and Conington et al. (2008) reported that increases in the incidence of mastitis could be halted by the inclusion of SCC in dairy cow genetic evaluations. Selection for resistance to mastitis in the French Lacaune dairy sheep breed is being attempted by the use of SCC as a proxy trait for mastitis and incorporating it into breeding programs (Rupp et al., 2002).
The detection of the ovine genome regions involved in mastitis resistance began with the first results of a Quantitative Trait Loci (QTL) detection program. It showed that QTLs for SCC were detected on chromosome 6 and 16, allowing locating the gene(s) that control resistance to mastitis in this species. Swiderek et al. (2006) described a possible correlation between Toll-like receptor (TLR)-gene mutations and the susceptibility of the mammary gland to bacterial infections. This study also showed the associate breed-dependent aspects of somatic cell concentration (SCC), bacterial infection and TLR-gene mutations in sheep. The data may serve as a benchmark for further study of TLR-gene mutation and may help in identifying one of the markers of natural resistance against sheep mastitis.
7. Economic consequences of mastitis
All partners involved with dairy ewes are increasingly concerned about the economic impact of mastitis (sheep farmers, veterinarians, dairy industry etc.). Adverse effects include death of the ewe, replacement, veterinary costs, reduced productivity, low weaning weights, deaths of lambs, degraded milk quality and impaired fertility of the affected ewes. Diseases of the udder are one of the most important reasons for premature culling and replacement of the ewes. Watson and Buswell (1984) observed that the percentage rises to 46% while Herrtage et al. (1974) raise the rate up to 50%. Severe depletion of milk production may lead to suboptimal growth of lambs and/or starvation and death of them. According to Menzies (2000) reductions in milk production have been reported to be between 20 and 37 per cent. That led at weaning to lambs from ewes with mastitis weighing up to 4 kg less than lambs from uninfected ewes. In another study, Conington et al. (2008) have found that the reduction in milk quality as a consequence of both clinical and subclinical mastitis led to a reduction in the rate of growth of the lambs of 66 g per day. The differences in milk production by affected and non-affected udders in sheep have been estimated to be up to 37 per cent (Fthenakis and Jones 1990), 50 per cent (Keisler et al., 1992) and 55 per cent (Saratsis et al. 1998). Barillet et al., (2001) mention that in french dairy sheep, the aversive effects of having milk with a high SCC resulted to economic losses of around 10 per cent. The control of mastitis in breeding sheep flocks must be organized in the way needed to establish the avoidable losses. To this direction, Yalcin et al. (1999) provided a modeling approach, which has been used in dairy cattles, in order to explore the possible impacts that mastitis may have on the financial performance of the flock.
Mastitis is an important and highly multivariable disease of sheep and presents varying degrees of duration, intensity and consequences. Studies confirm the trends observed of a significant increase in SCC and reduction in milk yield related to IMI (Luengo et al. 2004; Leitner et al. 2008). The characteristics of the IMI in conjunction with their breeding particularities; emerge the need to establish specific mastitis control programs in dairy ewes. This control plans, in order to assure udder health and high efficiency of production must focus in the control of milking routine and hygiene. Hence, the negative impact of environmental and management factors are minimized. During the past years much more information has become available on etiology, descriptive epizootiology, SCC, drying-off antibiotherapy. On the other hand, several aspects can be considered as still being poorly and further investigations should be performed in order to tackle the disease. There is, therefore, a definite need for further investigations about the genetics, incidence and financial consequences of mastitis in dairy ewes breeds.