Prevalence and aetiology of sheep mastitis in Alentejo region of Portugal

Small Ruminant Research 153 (2017) 123–130

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Prevalence and aetiology of sheep mastitis in Alentejo region of Portugal

MARK

M.C. Queiroga Departamento de Medicina Veterinária e Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal

A R T I C L E I N F O

A B S T R A C T

Keywords: Sheep mastitis Prevalence Aetiology California mastitis test Intramammary infection

Mastitis in ewes is responsible for massive economic expenses. Other than the loss of ewes due to clinical mastitis, when subclinical mastitis occurs, because it is not readily detected, the milk with the causative pathogens is used to make dairy products. This fact is disadvantageous both for the technological process and for the consumer security. The knowledge of prevalence and aetiological agents of mastitis is of outmost importance to carry out an effective control of mastitis in sheep. The aim of this work is to bring out ovine mastitis information in Portugal. We conducted a study focused on 18 milking producing sheep flocks to determine the prevalence of clinical and subclinical mastitis and to assess the aetiology of mastitis in sheep. Four hundred and fourteen milk samples randomly collected from sheep with subclinical mastitis and 27 milk samples from animals with clinical mastitis were bacteriological analysed according to an adaptation of the National Mastitis Council methodology. Anaerobic bacteria were also assessed. The identification was processed based on biochemical characterization, using the API system, API Staph, API 20 Strep, API 20 E, API 20 NE and API Coryne (BioMérieux). The results revealed high subclinical mastitis (SCM) prevalence (32.2%) and low clinical mastitis (CM) prevalence (1.7%). There was a higher prevalence in mechanically milked sheep than in hand milked animals. Coagulase negative Staphylococci (CNS) were in account of 42.9% of the CM events. Staphylococcus epidermidis represented 25% of all CM causative agents. Regarding subclinical mastitis, 70.1% of aetiological agents were identified as CNS species. From these, 43.4% were identified as Staphylococcus epidermidis accounting for 30.4% of total aetiological agents of SCM. Staphylococcus aureus was isolated from 22 specimens (6.2%) from SCM cases, which is of outmost importance in terms of public health, and Streptococcus agalactiae was isolated 15 times (4.2%) and showed to be in account of subclinical outbreaks with high prevalence rates. The fact that the majority of mastitis events were due to coagulate negative staphylococci out stresses the need for a proper management and rigorous hygienic milking procedure.

1. Introduction Mastitis in ewes is responsible for massive economic losses due to the death and early culling of animals (Arsenault et al., 2008; Jones, 1991), lower productivity (Dario et al., 1996; Gonzalo et al., 2002; Martí de Olives et al., 2013; Rovai et al., 2015) decreased lamb growth and survival (Fthenakis and Jones, 1990a) and deficient quality of the produced milk (Leitner et al., 2004 Quintana and Martín, 2005). Because subclinical mastitis is asymptomatic, getting unnoticed, the milk from affected animals is largely used for cheese production resulting in bad technological results, namely lower cheese production (Leitner et al., 2008; Rossi et al., 1994; Rovai et al., 2015; Silanikove et al., 2005) and reduced product quality (Wendorff, 2002). In Portugal, the production of sheep milk has been reaching major importance in the later years, as traditional cheese is getting vast

E-mail addresses: [email protected], [email protected]. http://dx.doi.org/10.1016/j.smallrumres.2017.06.003 Received 27 January 2017; Received in revised form 23 May 2017; Accepted 1 June 2017 0921-4488/ © 2017 Elsevier B.V. All rights reserved.

attention of consumers. Nevertheless, although the prevalence and aetiology of mastitis have been thoroughly investigated in cattle, this aspect has been somewhat neglected in ovine pathology and there have been no reports on ewe’s mastitis in this country. The aim of this study was to access the prevalence and aetiology of sheep mastitis in Évora, a district of Alentejo region, thus we conducted a bacteriologic survey in 18 dairy sheep flocks. 2. Materials and methods 2.1. Flocks and animals This study focused on 18 sheep flocks used for milk production, kept in extensive holdings in the district of Évora, Alentejo. The flocks comprised pure breeds, Awasii Lacaunne, German Merino, Spanish

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index was calculated for the different scores (“traces”, 1+, 2+ and 3+), as the highest Youden index should be the cut-off point (Redetzky et al., 2005).

Merino, White Regional Merino, Black Regional Merino, White Serra da Estrela, Black Serra da Estrela and cross breads of Awasii with Saloio, Regional Merino and other breads. In 5 farms milking procedure was hand milking and the remaining 13 flocks were mechanical milked.

Youden index = S + Sp − 1

2.2. Sample collection 3. Results

All mammary halves with clinical mastitis (CM) were acknowledged based on udder inflammatory symptoms and/or microscopic anomalous milk secretion. Detection of Subclinical Mastitis (SCM) was performed, through the California mastitis test (CMT), to a representative sample of animals chosen at random within each flock, calculated using the program “Win Episcope 2.0” based on the number of lactating sheep in each flock. As the prevalence was unknown, 50% prevalence was assumed, with a 95% confidence level with a 10% margin of error. From 4082 lactating sheep 1868 were submitted to CMT and, according to the obtained reaction, the result was recorded as negative, “traces”, 1+, 2+ and 3+. Ewes were considered to be affected with SCM if one or both mammary halves showed CMT result “traces”, 1+, 2+ or 3+. Twenty-seven milk samples were collected from animals with CM and 414 samples were randomly collected from sheep with SCM. Each milk sample was aseptically collected for a sterile bottle, after the teat was carefully disinfected with 70% ethanol and the first flush was rejected. Milk samples were kept refrigerated until they were processed, always in the same day of collection.

Clinical mastitis was detected in 36 animals out of 4082 expressing an average point prevalence of 0.9%, ranging from 0% to 8.1%. For hand milked flocks CM prevalence ranged from 0% to 2% with 0.2% average prevalence and mechanical milked animals exhibited CM prevalence 0–8.1% average 1.4%. Subclinical mastitis ranged from 1% to 92.5% with an average prevalence of 32.2%, when considering ewes, and 0.5% to 81.3% averaging 24,8% in the case of glands. Unilateral mastitis affected 48.9% of the ewes and bilateral mastitis reached 51.1%. The prevalence of SCM was significantly higher in flocks with mechanical milking than in those with hand milking (P < 0.0001). Mechanical milked flocks showed an average SCM prevalence of 42.6%, ranging from 7.8% to 92.5% with 33.1% gland’s prevalence. Regarding hand milked animals, the average SCM prevalence was 3.5% ranging from 1.0% to 8.0% with 2,2% gland’s prevalence. Concerning mastitis aetiology, out of the 27 milk samples collected from glands affected with CM, five resulted negative and 28 bacterial isolates were recovered from the remaining 22 samples. Twelve (42.9%) were identified as coagulase negative Staphylococci (CNS) (Table 1), of which 4 were present in mixed cultures (Table 2). From the 12 CNS isolates, 7 (25% of total aetiological agents) were identified as Staphylococcus epidermidis, only 2 of which were found in mixed cultures with Staphylococcus aureus and Streptococcus equinus (bovis), respectively (Table 2). Five isolates (17.9%) were identified as Staphylococcus aureus. Trueperella pyogenes was isolated 4 times (14.3%), only one of which in mixed culture (Table 2). Mixed cultures resulted from 6 milk samples collected from ewes with clinical mastitis (Table 2). As to microorganisms isolated from subclinical mastitis, 355 bacterial isolates were recovered from milk samples. The number of isolates of each bacterial species and respective percentages is shown in Table 3. Two hundred and forty nine isolates (70.1% of subclinical mastitis aetiological agents) were identified as CNS species. From these, 108 were identified as Staphylococcus epidermidis. Staphylococcus aureus and Streptococcus agalactiae were isolated 22 times (6.2%) and 15 times (4.2%), respectively (Table 3). Table 4 shows bacterial associations that were recovered from

2.3. Bacteriological analysis Bacteriological analyses were performed according to an adaptation of the National Mastitis Council methodology (National Mastitis Council, 2004). After shacked in a vortex mixer, 0.01 mL from each sample was plated on Blood Agar Base n° 2 (Oxoid, CM271) supplemented with 5% defibrinated ovine blood and 0.01 mL was plated on MacConkey Agar n° 3 (Oxoid, CM115). The plates were aerobically incubated at 37 °C for 24 h, followed by a further incubation in the same conditions if no growth was obtained after the first period. For anaerobic bacteria assessment, 0.01 mL of milk was plated on WilkinsChalgren Anaerobe Agar (Oxoid, CM619) and the plates were incubated in anaerobic jars (Anaerocult A, Merck, 1.13829.0001), at 37 °C, during 48 h. This study did not comprise Mycoplasma spp. detection. After incubation period, plates were examined for bacterial growths and all the colonies that grew in a number equal or superior to five were isolated for identification. The identification was processed based on morphological and Gram staining behaviour and biochemical characterization, using the API system: API Staph, API 20 Strep, API 20 E, API 20 NE and API Coryne (BioMérieux).

Table 1 Bacterial isolates from milk samples of ewes with clinical mastitis.

2.4. Statistical analysis

Bacteria

To assess the effect of mechanical and hand milking procedure on mastitis prevalence, data from 15 flocks (four hand and 11 mechanical), including 1749 sheep, were analysed by ANOVA using SAS Proc GLM with the following model:

Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus

Yij = μ + Oi + e

ij

epidermidis xylosus hyicus simulans lentus sp.

CNS Staphylococcus aureus Streptococcus agalactiae Streptococcus dysgslsctiae Streptococcus equinus (bovis) Granulicatella adiacens Trueperella pyogenes Pseudomonas aeruginosa Proteus sp. Total

Y – mastitis occurrence in sheep j submitted to milking procedure Oi μ – mean Oi – effect of milking procedure Oi eij – error To evaluate the use of CMT as an indicator of intramammary infection (IMI) in sheep and determine the cut-off point, sensibility (S), specificity (Sp), positive predictive value (pPV) and negative predictive value (nPV) were determined according to Thrusfield (1999). Youden 124

Total

% (total)

7 1 1 1 1 1

25.0 3.6 3.6 3.6 3.6 3.6

12 5 2 1 1 1 4 1 1 28

42.9 17.9 7.1 3.6 3.6 3.6 14.3 3.6 3.6 100

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Table 2 Bacterial isolates in mixed cultures from milk samples of ewes with clinical mastitis. Bacterial associations

Total

Staph. aureus + Staph. epidermidis Staph. aureus + Staph. hyicus Staph. aureus + Strep. dysgalactiae Staph. epidermidis + Strep. equinus Staph. xylosus + Trueperella pyogenes Strep. agalactiae + Granulicatella adiacens Total of mixed infections

1 1 1 1 1 1 6

Table 4 Bacterial isolates in mixed cultures from milk samples of ewes with subclinical mastitis.

Table 3 Bacterial isolates from milk samples of ewes with subclinical mastitis. Bacteria Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus

epidermidis xylosus hyicus simulans chromogenes warneri lentus capitis sciuri cohnii hominis caprae sp.

CNS Staphylococcus aureus Staphylococcus intermedius Micrococcus luteus Streptococcus agalactiae Streptococcus equinus (bovis) Streptococcus acidominimus Streptococcus equi zooepidemicus Streptococcus mitis Streptococcus suis Streptococcus sp. Enterococcus faecalis Enterococcus faecium Enterococcus durans Aerococcus viridans Lactococcus lactis Trueperella pyogenes Corynebacterium jeikeium Corynebacterium glucoronolyticum Corynebacterium accolens Corynebacterium striatum/amycolatum Corynebacterium sp. Pseudomonas aeruginosa Mannheimia haemolytica Pasteurella multocida Escherichia coli Serratia marcescens Enterobacter sp. Total

TOTAL

% (total)

% (CNS)

108 58 23 23 10 6 6 4 3 2 2 2 2

30.4 16.3 6.5 6.5 2.8 1.7 1.7 1.1 0.8 0.6 0.6 0.6 0.6

43.4 23.3 9.2 9.2 4.0 2.4 2.4 1.6 1.2 0.8 0.8 0.8 0.8

249 22 1 1 15 3 2 1 1 1 1 12 2 2 5 2 2 5 6 1 2 4 8 1 1 2 2 1 355

70.1 6.2 0.3 0.3 4.2 0.8 0.6 0.3 0.3 0.3 0.3 3.4 0.6 0.6 1.4 0.6 0.6 1.4 1.7 0.3 0.6 1.1 2.3 0.3 0.3 0.6 0.6 0.3 100

100

Bacterial associations

Total

Staph. aureus + Staph. xylosus Staph. aureus + Staph. chromogenes Staph. aureus + Strep. equinus (bovis) Staph. aureus + C. glucoronolyticum Staph. epidermidis + Staph. xylosus Staph. epidermidis + Staph. hyicus Staph. epidermidis + Staph. simulans Staph. epidermidis + Staph. warneri Staph. epidermidis + Staph. lentus Staph. epidermidis + Staph. capitis Staph. epidermidis + Strep. acidominimus Staph. epidermidis + Strep. mitis Staph. epidermidis + Enterococcus faecalis Staph. epidermidis + E. coli Staph. epidermidis + C. jeikeium Staph. xylosus + Staph. hyicus Staph. xylosus + Staph. chromogenes Staph. xylosus + Staph. warneri Staph. xylosus + Staph. cohnii Staph. xylosus + Staph. hominis + Corynebacterium sp. Staph. xylosus + Staph. caprae Staph. xylosus + Staph. sp. Staph. xylosus + Strep. sp. Staph. xylosus + C. glucoronolyticum Staph. xylosus + C. accolens Staph. xylosus + Corynebacterium sp. Staph. xylosus + Serratia marcescens Staph. hyicus + Staph. simulans Staph. hyicus + Staph. sp. Staph. simulans + Strep. acidominimus Staph. lentus + Staph. sciuri Staph. lentus + C. jeikeium + C. glucoronolyticum Staph. sciuri + Aeroc. viridans + C. glucoronolyticum Enterococcus faecalis + Aerococcus viridans C. glucoronolyticum + C. striatum/amycolatum Total of mixed infections

1 1 2 1 5 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 42

Table 5 Relationship between aetiological agent and mastitis magnitude. Mastitis reaction Isolate

Staph. epidermidis Staph. xylosus Staph. hyicus Staph. simulans Staph. chromogenes Staph. warneri Staph. aureus Strept. agalactiae Enterococcus spp. Aerococcus viridans Trueperella pyogenes Corynebacterium spp. Pseudom. aeruginosa

mammary glands with subclinical mastitis. The whole cultures for anaerobic bacteria resulted negative. It was possible to establish the relationship between the aetiological agent and the inflammatory reaction magnitude, relating the different grades of CMT and CM to the microorganism identified (N = 259). Only the mammary glands from which bacterial analysis disclosed pure cultures were considered (Table 5). The pattern of mastitis intensity produced by different microorganisms is shown in Fig. 1. Trueperella pyogenes infection induced the most expressive reaction, followed by Staphylococcus aureus. Regarding the utility of CMT to estimate IMI, bacterial cultures were positive from 53.1% milk samples with CMT score “traces”, 82.5% from

N

96 39 18 21 8 5 18 16 14 3 5 7 9

Trace

1+

2+

N

%

N

%

N

%

N

%

N

%

6 4 2

6,3 10,3 11,1

22 13 6 5

22,9 33,3 33,3 23,8

28,1 30,8 16,7 33,3 37,5 40 22,2 31,3 37,7

37,5 25,6 38,9 38,1 62,5 20 66,7 37,5 21,4 100 40 14,3 33,3

5

5,2

1

4,8

2 1

11,1 6,3

3

60

14,3 33,3

36 10 7 8 5 1 12 6 3 3 2 1 3

1

11,1

1

20

1

20

1 2

6,3 14,3

3 4

18,8 28,6

27 12 3 7 3 2 4 5 5

1 1

14,3 11,1

4 1

57,1 11,1

1 3

3+

CM

samples score 1+, 72.0% from samples score 2+ and 85.8% milk samples soring CMT 3+ (Table 6). Sensibility and Specificity considering different grades of CMT for cut-off are shown in Table 7. The highest Youden index (0.4) was obtained for CMT grade 1+.

4. Discussion Our study revealed an average clinical mastitis point prevalence of 0.9%, ranging from 0% to 8.1%. Other authors state higher prevalence 125

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Fig. 1. Pattern of mastitis intensity produced by different microorganisms.

Staph. epidermidis Staph. xylosus Staph. hyicus Staph. simulans Staph. chromogenes Staph. warneri Staph. aureus Strep. agalactiae Enteroc. spp. Aeroc. viridans Truep. pyogenes Coryn. spp. Pseud. aeruginosa 0%

20%

40% T

1+

60% 2+

3+

80%

MC

genotype, the stage of lactation and milking management should also be considered. Other references to the effect of mechanical and hand milking procedure on the prevalence of mastitis are contradictory: Some authors, in accordance with the present results, refer to somatic cell counts in ewes milked mechanically superior to counts obtained from hand milked (Marguet et al., 2000); others did not find differences between mechanical and hand milking procedure (González-Rodríguez et al., 1995) and some others mentioned a lower mastitis prevalence with mechanical milking ewes than those with hand milking (Las Heras et al., 1999b; Marogna et al., 2010). According to Mavrogianni et al. (2006), hand milking does not significantly increases the risk of intramammary infection in ewes. However, mechanical procedure may adversely affect the teat tissues, reducing local defence mechanisms, causing greater risk of infection than hand milking (Hamann and Stanitzke, 1990; Zecconi and Hamann, 2005), and it may even affect the teat sphincter in the long term (Rainard and Riollet, 2006). Milking equipment may be responsible for the transmission of microorganisms from one animal to another, which may be passive transmission when these agents are deposited near the teat opening, or active transmission when bacteria are forced into the mammary gland by the machine itself. In addition, the malfunction or careless use of the milking machine can cause injuries that favour infection (Peris et al., 2001). Possibly mechanical milking of ewes in Portugal is still not sufficiently adjusted to species specificities. In fact, at the beginning of dairy cattle mechanical milking, the incidence and prevalence of MSC was very high (Henriques, 1969) and only later, with the improvement of devices and the rectification of management errors, was possible to decrease those rates (Sampimon et al., 2005). Regarding aetiology, our results indicated a relevant responsibility of coagulase negative Staphylococci for ovine mastitis, representing 42.9% of clinical mastitis aetiological agents and 70.1% of bacteria accountable for subclinical mastitis. Coagulate negative Staphylococci have been mentioned as responsible for subclinical mastitis in sheep in different countries, namely Spain (De la Cruz et al., 1994; González-Rodríguez et al., 1995; Las Heras et al., 1999b; Marco et al., 1993; Ziluaga et al., 1998), Italy (Cuccuru et al., 2011; Dario et al., 1996; Dore et al., 2016), Greece (Fthenakis et al., 2004; Kiossis et al., 2007; Stefanakis et al., 1995), France (Bergonier et al., 2005b), U. K. (Hariharan et al., 2004), U. S. A. (Spanu et al., 2011) and Israel (Leitner et al., 2001). Staphylococcus epidermidis was in account of 25% of CM cases and was isolated from 30.4% of the samples from glands with SCM. This species represented 43.4% of CNS isolated from SCM events.

Table 6 Relationship between CMT results and bacterial cultures. CMT

N V + ++ +++

Bacterial cultures

10 64 97 118 134

Positive culture

Negative culture

n

%

n

%

0 34 80 85 115

0 53,1 82,5 72,0 85,8

10 30 17 33 19

100 46,9 17,5 28,0 14,2

Table 7 Sensibility and specificity of CMT for different cut-off points. Cut-off

Sensibility (S)

Specificity (Sp)

pPV

nPV

V + ++ +++

1 0,7 0,43 0,37

0,25 0,7 0,63 0,83

0,53 0,82 0,72 0,86

1 0,54 0,33 0,31

100%

rates, 19% to 22%, but they often refer to outbreaks reports (Las Heras et al., 1999a; 2002; Radostists et al., 2000) or to annual average prevalence, 3.1% (Bergonier et al., 2005a). Concerning subclinical mastitis prevalence, our work disclosed quite a diversity of results in different flocks, ranging from 1% to 92.5%, getting an average prevalence of 32.2%, regarding ewes, and an average gland prevalence of 24.8%, ranging from 0.5% to 81.3%. Other studies refer ewes’ prevalence rates between 6.4% and 83% and gland prevalence ranging from 3.1% to 67% (Ahmad et al., 1992; Bio et al., 2005; Bor et al., 1989; Dario et al., 1996; De la Cruz et al., 1994; González-Rodríguez et al., 1995; Jones, 1991; Las Heras et al., 1999b, 1999c; Marco et al., 1993; Pengov, 2001; Romeo et al., 1998; Stefanakis et al., 1995; Watkins et al., 1991; Watson et al., 1990; Ziluaga et al., 1998). This multiplicity of results is probably due to different methodological approaches of SCM diagnosis. The results obtained in this study showed mastitis prevalence far superior in flocks mechanically milked than in the hand milked ones. This difference was found for MC prevalence but was more pronounced for MSC prevalence. However, the highly significant difference found between the two milking procedures should be carefully analysed, due to the great variability of prevalence rates found in various mechanical milked flocks. The lack of information available with respect to sheep 126

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et al., 2010; Ziluaga et al., 1998); Streptococcus equi subsp. zooepidemicus, mentioned as the cause of a CM outbreak in sheep (Las Heras et al., 2002); Streptococcus suis, also isolated by Marogna et al. (2010), and Streptococcus mitis, from which we did not find bibliographic references relating them to ovine mastitis. Enterococci species accounted for 4.5% of the cases of subclinical mastitis. The following species were found: Enterococcus faecalis and Enterococcus faecium, which were previously isolated from SCM (Watkins et al., 1991) and CM in ewes (Marogna et al., 2010) and Enterococcus durans that was also reported by Marogna et al. (2010) as CM aetiological agent. Aerococcus viridans was isolated from 5 milk samples from sheep with subclinical mastitis belonging to 3 different flocks. Its association to ovine mastitis was reported before (Ziluaga et al., 1998). Corynebacteria were recovered from 18 cases (5%) of subclinical mastitis. The species identified were Corynebacterium jeikeium, C. glucoronolyticum, C. accolens and C. striatum or C. amycolatum, as the API Coryne method does not discriminate these two species. Other corynebacteria species have been recovered from specimens of ewe subclinical mastitis, namely C. mastitidis (Fernández-Garayzábal et al., 1997) and C. camporealensis (Fernández-Garayzábel et al., 1998). The API Coryne database does not include these species. Frequently allusions to corynebacteria associated with subclinical mastitis in sheep are referred as to Corynebacterium spp. (Apolo et al., 1999; De la Cruz et al., 1994; Gelasakis et al., 2015; Mavrogenis et al., 1995; Stefanakis et al., 1995; Watson et al., 1990; Ziluaga et al., 1998), perhaps because it is not easy to succeed with the species identification. These details taken together with the fact that in the present study four different species were identified, which haven’t previously been isolated from ovine mammary gland, suggest the misidentification of these microorganisms. Other gram-positive bacteria were identified as ovine mastitis aetiological agents in the present study as well as in previous works. These were Staphylococcus intermedius (Las Heras et al., 1999c), Micrococcus luteus (De la Cruz et al., 1994) and Lactococcus lactis (Pengov, 2001). Only two cases of CM were caused by Gram-negative bacteria: Proteus sp., that have previously been in account of subclinical mastitis in ewes (Burriel, 1997; Gelasakis et al., 2015; Las Heras et al., 1999b, 1999c; Ziluaga et al., 1998) and Pseudomonas aeruginosa. The later was also responsible for 8 cases (2.3%) of subclinical mastitis. This microorganism is frequently reported as to clinical (Falade et al., 1988; Las Heras et al., 1999a; Rapoport et al., 1999; Watson et al., 1990) and subclinical mastitis causative agent in sheep (Bor et al., 1989; GonzálezRodríguez et al., 1995; Rapoport et al., 1999). Other Gram-negative bacteria in account of subclinical mastitis were Mannheimia haemolytica, Pasteurella multocida, Escherichia coli, Serratia marcescens and Enterobacter sp. All of which have been previously referred as ewe subclinical mastitis pathogens (Apolo et al., 1999; Bor et al., 1989; Dario et al., 1996; Gelasakis et al., 2015; González-Rodríguez et al., 1995; Mavrogenis et al., 1995; Rapoport et al., 1999; Watkins et al., 1991; Watson et al., 1990; Ziluaga et al., 1998). Mixed infections occurred in 42 cases of subclinical mastitis and in 6 cases of clinical mastitis. The association of Staphylococcus epidermidis with Staphylococcus xylosus occurred 5 times. These two species were the most frequently isolated species during the study. Only 3 other combinations occurred more than once. A relationship between the etiological agent and inflammation intensity was detected. The inflammation induced in the mammary gland in response to infection is responsible for the increased somatic cells discharge, which are mainly neutrophils. The CMT score is greater the higher the number of cells present in milk. The results of this study suggest different inflammatory response profiles for various species of microorganisms. Among the etiological agents isolated in this study, Trueperella pyogenes induced the most expressive reaction. In contrast, the inflammatory pattern driven by Corynebacterium spp. was the most

Staphylococcus epidermidis has been mentioned by several researchers as the main aetiological agent of ewes SCM (Ariznabarreta et al., 2002; Bergonier et al., 2005b; Cuccuru et al., 2011; De la Cruz et al., 1994; Hofer et al., 1995; Kiossis et al., 2007; Las Heras et al., 1999b, 1999c; Pengov, 2001; Ziluaga et al., 1998) and was reported by Hofer et al. (1995) as the main cause of clinical mastitis in sheep and by Marogna et al. (2010) as the second in prevalence, after Streptococcus uberis. This species also causes the majority of persistent infections of ovine mammary gland (Bergonier et al., 2005a). Staphylococcus aureus has been considered the major causative agent of ewe’s clinical mastitis (Bergonier et al., 1999; Falade et al., 1988; Gutierrez et al., 1990; Indrebø, 1990; Jones, 1991; Mavrogenis et al., 1995; Mørk et al., 2007; Radostists et al., 2000). These citations, though, refer to mastitis outbreaks with high incidence of affected animals that show severe symptoms, which become noticed by the sheep owner. In the present study only 5 milk samples (17.9%) from mammary glands affected with clinical mastitis revealed the presence of Staphylococcus aureus, 3 of which were in mixed cultures. These differences may be due to the reduced number of animals with clinical mastitis analysed, or to the disparity of geographic and climatic factors that interfere with the microbial population. The fact that 5 out of 27 analysed samples have resulted negative suggests the hypothesis of intracellular localization of the causative agent (Almeida et al., 1996; Almeida and Oliver, 2001). Concerning subclinical mastitis, Staphylococcus aureus was isolated from 22 (6.2%) milk samples, but this species reached 12.6% of subclinical mastitis aetiological agents in one of the studied flocks. This fact is of outmost importance in terms of public health, as Staphylococcus aureus is often thermo stable enterotoxin producer and, because SCM is generally not detected, the milk from affected ewes is used for cheese production. Prevalence of SCM due to Staphylococcus aureus is usually lower in ewes (De la Cruz et al., 1994; Las Heras et al., 1999b; Leitner et al., 2001; Marco et al., 1993; Pengov, 2001; Watkins et al., 1991; Ziluaga et al., 1998) than in cows (Bexiga et al., 2005; Bolzoni et al., 2005). This may be due to the removal of most virulent stains from the flocks as the affected animals are culled, as suggested by Leitner et al. (2001). Also the high prevalence of intramammary infections due to CNS may hinder the colonization by Staphylococcus aureus (Woodward et al., 1987, 1988). Trueperella pyogenes was isolated from 4 clinical mastitis cases and from 2 subclinical mastitis samples. The association of this microorganism with sheep mastitis has been mentioned, both for clinical (Falade et al., 1988) and subclinical cases (Jones, 1991; Rapoport et al., 1999). Streptococcus agalactiae was recovered from 2 cases of clinical mastitis and was responsible for 15 cases of subclinical mastitis. Even though is not frequently isolated from ovine intramammary infections, it may cause clinical mastitis (Falade et al., 1988) and it may be the source of subclinical outbreaks. It was the aetiological agent of 40% of subclinical cases in one of the flocks under this study and was previously reported to have been isolated from 13.5% to 31% of sheep with subclinical mastitis (Bio et al., 2005; Las Heras et al., 1999b, 1999c). Streptococcus equinus (bovis) was isolated from 1 case of clinical mastitis in association with Staphylococcus epidermidis and from 3 cases of subclinical mastitis, one of which in pure culture. This microorganism has been reported as the cause of sheep CM (Marogna et al., 2010) and subclinical mastitis that evolved to the clinical form (Watkins et al., 1991). Streptococcus dysgalactiae was identified once from a clinical mastitis case in association with Staphylococcus aureus. Its association with ovine mastitis has been referred (Bor et al., 1989; Marogna et al., 2010; Scott, 2000). Granulicatella (Streptococcus) adiacens was also recovered in association from a case of clinical mastitis, but we could not find any reference to its connection to ovine mammary pathology. Other streptococci species related to subclinical mastitis were Streptococcus acidominimus, which was previously reported (Marogna 127

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sheep producer objective. The more demanding is the criteria and more ambitious, in terms of udder health, the lower should be the cut-off. The results of this study showed that the use of reaction “traces” as cut-off can eliminate the false negative cases, as the negative predictive value was 1. However, the percentage of false positives was 46.9% and the positive predictive value was 0.53.

Table 8 Comparison of our results with other authors data, ordering CNS according to the intensity of mammary gland inflammation. CNS

Staph. Staph. Staph. Staph. Staph. Staph.

chromogenes simulans epidermidis hyicus xylosus warneri

Our results

Fthenakis and Jones (1990b)

1° 2° 3° 4° 5° 6°

1° 2°

Burriel (1997)

Ziluaga et al. (1998)

2° 3°

2° 1° 4°

1°

5° 3°

3°

Pengov (2001)

5. Conclusion

3° 1° 2°

From these results stresses out the occurrence of ovine mastitis caused by contagious microorganisms, namely Staphylococcus aureus that is of outmost significance in terms of public health and Streptococcus agalactiae that may be in account of subclinical outbreaks with high prevalence rates. Not withstanding, the majority of mastitis events were due to coagulate negative staphylococci. These bacteria integrate the skin microbiota, which out stresses the need for proper management and rigorous hygienic milking procedure.

moderate, confirming the results of other studies, which reported that the Corynebacterium species are associated with discrete elevations of somatic cell count (Ziluaga et al., 1998). Staphylococcus aureus induced intensive inflammation shown by MC episodes and MSC with high levels of somatic cells in milk producing CMT reactions of 2+ and 3+. Sheep intramammary infections caused by this pathogen induces high somatic cell count (González-Rodríguez et al., 1995; Ziluaga et al., 1998). The pattern of CMT reactions of milk from infected glands with Streptococcus agalactiae and Pseudomonas aeruginosa are relatively similar and, as mentioned by González-Rodríguez et al. (1995), the cellular response to intramammary infections due to these microorganisms is pronounced. When different species of SCN are studied separately and compared to each other, different results are stated. Some studies refer to similar inflammatory reactions for different species (Leitner et al., 2001) but others describe variable effects according to the species. However, the various references refer to different response produced by each species (Burriel, 1997; Fthenakis and Jones, 1990b; Pengov, 2001; Ziluaga et al., 1998). According to the inflammatory intensity profiles, the different species of SCN isolated in this study could be ordered, starting with the species that elicited the greater degree of inflammation, as follows: Staph. chromogenes → Staph. simulans → Staph. epidermidis → Staph. hyicus → Staph. xylosus → Staph. warneri. Table 8 shows a comparison of the results obtained in this study and the results referred by other authors. Aerococcus viridans was isolated in pure culture only in 3 occasions, always inducing a CMT 3+ reaction. We could not find any reference to the influence of this microorganism on the inflammation of the mammary gland. With regard to the study on the use of the California mastitis test as an indicator of intramammary bacterial infection, the highest Youden index was reached with 1+ grade. Taking 1+ as cut-off, both sensitivity (S) and specificity (Sp) of the test will be 0.7. These values are low. When considering as negative the mammary glands whose milk reveals a CMT reaction below 1+, we get a high rate of false negatives. Indeed, 53.1% of the milk with “traces” reaction revealed intramammary infection. Las Heras et al. (1999c) suggest the use of CMT 1+ grade as a MSC indicator value in sheep, after obtaining 0.84 for S and 0.91 for Sp. Other authors got different S and Sp values for the same cut-off: 0.46 and 0.97, respectively, (De la Cruz et al., 1994); 0.73 and 0.83 (McDougall et al., 2001); 0.91 and 0,79 (Lafi, 2006). Romeo et al. (1998) determined S and Sp values for CMT cut-off 1+ in monthly collected samples throughout lactation, getting S values ranging from 0.29 to 0.60 and Sp values between 0.72 and 0.93. The disparity of values may be due to variations in test interpretation by different operators, as this is a highly subjective method. Despite CMT predictive values are not high, it is a low price and easy to perform test, which allows immediate diagnosis and therefore very useful for tracking udder health status by sheep producers. The cut-off choice depends on the

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