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The Veterinary Journal 176 (2008) 378–384

TheVeterinary Journal

Subclinical mastitis changes the patterns ofmaternal–offspring behaviour in dairy sheep

D.A. Gougoulis a, I. Kyriazakis a, N. Papaioannou b, E. Papadopoulos b,I.A. Taitzoglou b, G.C. Fthenakis a,*

a Veterinary Faculty, University of Thessaly, P.O. Box 199, 43100 Karditsa, Greeceb School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Macedonia, Greece

Accepted 28 February 2007

Abstract

Subclinical mastitis was induced by inoculating one mammary gland in dairy ewes (n = 8) with a Staphylococcus simulans isolate; con-trol ewes (n = 4) were included. The milk yield of inoculated glands decreased (P < 0.001), the California Mastitis Test (CMT) scoreincreased and the organism could be recovered from the inoculated glands. With time, there was significantly increased frequency of‘‘hindering sucking’’ (P = 0.016) and ‘‘head up posture’’ (P < 0.001) in the control ewes. Infected ewes had a significantly increased fre-quency of ‘‘vocalisation’’ (P = 0.013) compared to controls. There was a significant difference in the frequency of ‘‘sucking attempt’’ and‘‘successful suck’’ (P < 0.05) behaviours between lambs of the two groups. Lambs of the challenged ewes also showed significantlyincreased frequency and duration of these behaviours towards the uninoculated glands of their dams, rather than to the challengedglands (P < 0.05); no such difference was evident for the lambs of control ewes. It was concluded that subclinical mastitis alters the suck-ing behaviour of both ewes and lambs.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Sheep; Subclinical mastitis; Staphylococcus simulans; Sucking behaviour

1. Introduction

The importance of subclinical mastitis as a limiting fac-tor in milk production in ewes is well established: subclin-ical mastitis results in decreased milk production(Fthenakis and Jones, 1990a; Keisler et al., 1992; Saratsiset al., 1999) and growth retardation of lambs from affectedewes (Kalinowska et al., 1985; Fthenakis and Jones,1990a). Coagulase-negative staphylococci are consideredto be the primary causal agents of subclinical mastitis (Fth-enakis and Jones, 1990b; Las Heras et al., 1999; Bergonierand Berthelot, 2003).

Studies of lactational behaviour in sheep have estab-lished the development and patterns of sucking behaviour,as well as factors potentially affecting this behaviour (Poin-

1090-0233/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2007.02.024

* Corresponding author. Tel.: +30 24410 66070; fax: +30 24410 66077.E-mail address: gcf@vet.uth.gr (G.C. Fthenakis).

dron et al., 1993; Nowak, 1996; Dwyer, 2003). However,the health of the mammary gland, i.e., the possible presenceof mastitis, was not taken into account during these studiesand no samples of mammary secretions were examined forevidence of mastitis. Previous reports have suggested thatsubclinical mastitis might interfere with sucking behaviour(Fthenakis, 1988; Fthenakis and Jones, 1990a). Further-more, transmission of bacteria into the teats of ewes duringsucking has now been documented (Gougoulis et al., inpress). Hence, there is a possibility that microorganismsfrom ewes with mastitis may be transmitted to healthy ani-mals through ‘‘cross-sucking’’. This point is of particularimportance in dairy ewes, where ewes are milked for 3–4months after the sucking period.

Any case of mastitis during the suckling period will haveconsequences for subsequent milk production within thesame lactation. Therefore, there is scope to study epimeletic(care giving) and etepimeletic (care seeking) behaviour in

D.A. Gougoulis et al. / The Veterinary Journal 176 (2008) 378–384 379

cases of mastitis, with a view to evaluating possible devia-tions from established normal patterns of behaviour. Theobjective of the present work was to study whether ovinesubclinical mastitis affects dam and offspring behaviourduring lactation.

2. Materials and methods

2.1. Experimental overview

Eight primiparous Karagouniko-breed ewes, each rearing a singlelamb, were used in the study. Unilateral subclinical mastitis was inducedby direct intramammary inoculation with approximately 5 · 106 colony-forming-units (cfu) of Staphylococcus simulans (group I), which had beenisolated from a case of ovine clinical mastitis (Fthenakis, 1988). Anadditional four ewes were used as uninoculated controls (group C). Sub-clinical mastitis was defined by simultaneous bacterial isolation andpresence of an inflammatory reaction in the mammary gland, i.e., func-tional changes, in the absence of abnormal gross findings systemically orin the mammary gland (Schalm et al., 1971; Fthenakis and Jones, 1990a,1990b; Fthenakis, 1994; Bergonier and Berthelot, 2003; Bergonier et al.,2003).

Ewes were housed in individual straw-bedded pens (1.8 m · 5.0 m),allowing visual contact between adjoining animals, and given concentratefeed and hay. The animals were allocated into groups in such a way thatthe ratio of male and female lambs in a given group was the same and thatthe mean birthweight of lambs in each group was not significantly dif-ferent. Ewes were then inoculated on the eighth day after lambing (D0).The work was carried out under a licence for experimental proceduresobtained from the Greek Ministry of Agriculture.

2.2. Preparation of inocula and inoculation technique

S. simulans was grown on Columbia blood agar, checked for purityand inoculated into Soy-broth (BioMerieux) for aerobic incubation at37 �C for 5 h. Serial dilutions of the broth culture into phosphate bufferedsaline (PBS), were carried out to a final concentration of 5 · 106 cfu (Milesand Misra, 1938). A syringe was used to inoculate 0.2 mL of this sus-pension into the mammary gland by means of a 20 G fine sterile plasticcatheter (Abbocath, Abbott), inserted into the gland cistern of group Iewes. Four ewes were challenged in the right and four in the left mammarygland. The same technique was used to inject 0.2 mL of PBS into thecontralateral mammary gland of these ewes and into both mammaryglands of group C ewes.

2.3. Clinical and laboratory procedures

General clinical examination and mammary examination were carriedout on days D-5, D-3 and D-1 (before challenge, i.e., 2, 5 and 7 days afterlambing, respectively) and on D1, D3, D6, D11, D16, D21 and D26.Samples of mammary secretion were collected following established pro-tocols (Fthenakis, 1994; Mavrogianni et al., 2005).

Conventional bacteriological techniques were used throughout thestudy (Euzeby, 1997). Samples were plated onto 5% Columbia blood agarand incubated aerobically at 37 �C for up to 72 h. Staphylococcal isolateswere identified by means of the API-Staph SYSTEM quick identificationstrips (BioMerieux) and profiles of isolates were compared. The CaliforniaMastitis Test (CMT) was carried out on samples of secretion with fivereaction scores: negative, trace, 1, 2 and 3 (Fthenakis, 1995). Films ofsecretion were stained by the Giemsa method for identification of leuco-cytes subpopulations.

Milk yield measurements were carried out on D0 prior to challengeand then on D7, D17 and D27. Milk yield was estimated as described byFthenakis and Jones (1990a). Lambs were weighed on D-5 and on D0 andthen on D7, D17 and D27. Faecal samples were also collected from lambsfor parasitological examination. They were processed by using the zinc

sulphate flotation method, in order to detect coccidial oocysts and nem-atode eggs. Faecal smears were stained by the saffranin-methylene bluemethod to detect Cryptosporidium spp. oocysts.

Ewes were euthanased on D27 and detailed pathological examinationof the mammary glands (El-Masannat et al., 1991) was carried out. Tissuesamples were submitted for bacteriological examination as above. Tissuesections were processed for histological examination using conventionaltechniques.

2.4. Behavioural observations

Behavioural data were collected by two digital video cameras (Model92E, Sony) using a continuous focal observation method on individualewes and their lambs. The daily observation period was set from 0900 h to2100 h each day, with a 2 h interval in the afternoon. During the obser-vation period, no human movements within the pen area were allowed.

Behaviours were recorded on the same days as samples were collected.A daily observation schedule of 100 min for each ewe was undertaken.Observations were carried out in a pseudo-random manner to ensure thatdata collection for each ewe and her lamb was equally spaced within thetotal observation period. All behaviours of each ewe and her lamb wererecorded. Definitions of the ewe and lamb behaviours recorded during thestudy (‘‘ethogram’’) are given in Table 1.

2.5. Data management and analysis

2.5.1. Definitions

We divided lactation into three stages for calculations and breakdownof results: Stage 1: Before challenge; Stage 2: D1–D13; Stage 3: D14–D27.We also made the following calculations:

Mean milk yield ðmMYÞ on a sampling day

¼ ½MY1 þMY2 þ � � � þMYn�=8;

where MYi, milk collected from each gland of a set on that sampling. Weconsidered separately three sets of glands: challenged glands of group Iewes (n = 8); Control glands of group I ewes (n = 8); All glands of group Cewes (n = 8)

Total mean milk yield ðTmMYÞ after challenge

¼ ½mMYD7þmMYD17þmMYD27�;

where mMY, mean milk yield on each of D7, D17 or D27.

Mean lamb bodyweight ðmLWÞ ¼ ½LW1 þ LW2 þ � � � þ LWn�=½N �;

where LWi, bodyweight of each lamb in group; N, number of lambs ingroup

Mean daily growth rate of lambs ðmDGRÞ¼ ½ðLW1D27-LW1D0Þ=27� þ ½ðLW2D27-LW2D0Þ=27� þ � � �þ ½ðLWnD27-LWnD0Þ=27�=½N �;

where LWiD27, bodyweight of each lamb in group on D27; LWiD0, body-weight of each lamb in group on D0; N, number of lambs in group.

Frequency of a specific behaviour ðFbÞ per animal

¼ ½B1 þ B2 þ :::þ Bn�=½D�;

where Bi, number of events of that specific behaviour recorded during thetotal observation period within each stage of lactation; D, number ofobservation days within each stage of lactation.

Mean frequency of a specific behaviour ðmFbÞ¼ ½Fb1 þ Fb2 þ � � � þ Fbn�=½N �;

where Fbi, frequency of the specific behaviour per animal; N, number ofanimals in group.

Table 1Definitions of ewe and lamb behaviours recorded during focal observation (‘‘ethogram’’)

Definitions of ewe behaviours

Lie (LD) Lying down inactive or chewing or ruminatingHindering sucking (HC/

HB/HF)Moves as lamb approaches the udder attempting to suck; circling: moves hindquarters away from lamb (HC); backing:moves backwards away from lamb (HB); forwarding: steps forwards over the top of lamb (HF)

Head up posture (HP) Moves towards light and stands waiting for lamb(s) to suckVocalisation (VL/VH) Emits low-pitched, mouth closed (VL) or high-pitched, mouth open (VH) bleatsEating (E) Includes biting, chewing and searching for food

Definitions of lamb behaviours

Lie (LD) Lying down inactive or chewing or ruminatingSucking attempt (SA) Is under ewe in udder region, in an inverse parallel position to the damSuccessful suck (SS) Has teat into mouth and appears to suck, for >5 sSucking bout (SB) One SS which is >10 s after the previous one and before the next one or a sequence of at least two SS <11 s apartVocalisation (V) BleatsEating (E) Includes biting, chewing and searching for food

380 D.A. Gougoulis et al. / The Veterinary Journal 176 (2008) 378–384

Proportion of duration of a specific behaviour ðDbÞ¼ ½T1 þ T2 þ � � � þ Tn� � 100=½D� 100�;

where Ti, duration (min) of events of that specific behaviour recorded dur-ing the total observation period within each stage of lactation; D, numberof observation days within each stage of lactation.

Mean proportion of duration of a specific behaviour ðmDbÞ¼ ½Db1 þDb2 þ � � � þDbn�=½N �;

where Dbi, proportion of duration of the specific behaviour per observa-tion slot per animal (ewe or lamb); N, number of animals in group. Noduration was calculated for instantaneous behaviours, i.e., behaviourswhere each event typically lasted <2 s: ‘‘hindering sucking’’, ‘‘head up pos-ture’’ and ‘‘vocalisation’’.

Frequency and proportion of duration of ‘‘sucking attempt’’ and‘‘successful suck’’ were also calculated in relation to the mammary gland(left or right) sucked by the lamb on each occasion. For group I ewes,separate results for challenged (‘‘i’’) and control (‘‘c’’) glands were pro-duced. For group C ewes, two right and two left glands (‘‘p1’’) wereselected at random and considered together; the other two right and theother two left glands (‘‘p2’’) were also considered together.

2.5.2. Statistical methods

Proportions were compared by employing the v2-test. Paired data werecompared by using a two-sample t test. Analysis of co-Variance withrepeated measures taken as mean over time, was used for remainingcontinuous data; pre-challenge measurements were used as co-variate.Data were modelled by means of the General Linear Model applied inMinitab 14 (Minitab Inc.). Data were tested for normality; appropriatetransformations were performed where necessary. Statistical significancewas defined as P < 0.05.

3. Results

3.1. Clinical observations

All ewes lambed without obstetrical assistance; totallambing time never exceeded 2 h. The ewes duly cared fortheir lambs and no neonatology intervention was needed.Ewes remained clinically healthy and no abnormal systemicor mammary signs were recorded throughout the study.Teat bites were evident in the inoculated side of one groupI ewe but no teat bites were evident in group C ewes. Noabnormal findings were recorded in any lamb. All animalswere retained to the conclusion of the study.

3.2. Bacteriological and cytological findings

Prior to D0, no bacteria were isolated from any secre-tion sample. The CMT was positive (score 1) in only afew (15/72) samples. No leucocytes were observed inGiemsa-stained films from these samples.

Subclinical mastitis developed in all inoculated mam-mary glands. S. simulans was isolated consistently up toD16 and, in total, from 49/56 samples from inoculatedglands. The CMT increased (score P2) on D1 in all inoc-ulated glands and, in total, was positive in 51/56 samples.Leucocytes were seen in Giemsa-stained films of the secre-tions; initially, the great majority (>80%) of leucocytes con-sisted of neutrophils, with a few macrophages andlymphocytes (5–15%) also present; subsequently, the pro-portion of neutrophils decreased (30–70%), whilst that ofmacrophages and lymphocytes increased (0–30% and 10–50%, respectively).

Neither bacterial isolations, nor CMT positive (score6trace) results, were obtained from the control glands ofgroup I ewes, nor from any gland of group C ewes afterD0. No leucocytes were observed in Giemsa-stained filmsfrom these glands.

3.3. Milk yield

Prior to challenge, there was no significant differencebetween mMY of group I glands to-be-challenged, mMYof group I glands to-be-controls and mMY of group Call glands (P > 0.4) (Table 2). Subsequently, TmMY ofgroup I challenged glands was significantly reduced com-pared to that of group I control glands and of group Call glands (P < 0.001). There was no significant differencebetween TmMY of group I control glands and of groupC all glands (P = 0.194).

3.4. Lamb weights and parasitological examinations

There were no significant mLW differences among groupI and group C lambs at birth (D-5: mean weight 5.19 kgand 4.93 kg, respectively; P = 0.304), D0 (mean weight

Table 2Milk yield (mL) of ewes challenged by intramammary inoculation withS. simulans (group I) and controls (group C)

mMY TmMY (on D7, D17 andD27)

Group I ewes: challenged glands(n = 8)

141 ± 33* 148 ± 46a

Group I ewes: control glands(n = 8)

147 ± 26 529 ± 57b

Group I ewes: all glands (n = 16) 144 ± 29 338 ± 49c

Group C ewes: all glands (n = 8) 153 ± 37 593 ± 105b

mMY = [MY1 + MY2 + ÆÆÆ + MYn]/8, where MYi = Milk collected fromeach gland of a set on that sampling.TmMY = [mMYD7 + mMYD17 + mMYD27], where mMY = Meanmilk yield on each of D7, D17 or D27.a,b,c Different letters within the same column define statistically significantdifference (P < 0.05).

* Mean ± standard error of the mean.

D.A. Gougoulis et al. / The Veterinary Journal 176 (2008) 378–384 381

6.41 kg and 6.18 kg, respectively; P = 0.404) or D27 (meanweight 12.54 kg and 12.12 kg, respectively; P = 0.342). As aconsequence, there was no significant difference in mDGRamong these lambs (227 g and 220 g, respectively; P =0.814).

There were no differences in results of parasitologicalexaminations among lambs of the two groups. No diar-rhoea was recorded in any lamb. No nematode eggs orCryptosporidium spp. oocysts were found in faecal samples.Eimeria spp. oocysts were found; mean count (per g) on D0was 12.5 ± 8.2 for group I and 12.5 ± 12.5 for group C; onD27 it was 50.0 ± 9.4 and 75.0 ± 14, respectively.

Table 3Mean frequency of behaviours (mFb) of ewes challenged by intramammary ino

Behaviour abbreviation 1st Stage 2nd

I C I

Ewe behaviours

Lie 1.76 ± 0.19* 1.81 ± 0.24 1.7Hindering sucking 1.64 ± 0.36 0.73 ± 0.28a 3.0

Circling 0.25 ± 0.15 0.01 ± 0.01 0.1Backing 0.00 ± 0.00 0.00 ± 0.00 0.1Forwarding 1.17 ± 0.29 0.41 ± 0.28 2.1

Head up posture 1.00 ± 0.23 0.59 ± 0.22a 0.9Vocalisation 1.58 ± 1.02 1.08 ± 1.08a 0.8

Low-pitched 0.29 ± 0.30 0.75 ± 0.75 0.0High-pitched 0.38 ± 0.33 0.08 ± 0.08 0.1

Eating 4.41 ± 1.15 3.58 ± 1.57 4.4

Lamb behaviours

Lie 4.43 ± 0.06 4.35 ± 0.22 4.8Sucking attempt 11.38 ± 1.42 13.72 ± 1.42 10.9Successful suck 9.44 ± 1.40 12.18 ± 1.54 9.6Sucking bout 4.13 ± 0.47 4.57 ± 0.28 3.1Vocalisation 2.47 ± 0.62a 3.12 ± 0.00a 0.0Eating 0.55 ± 0.23a 0.47 ± 0.29a 3.5

mFb = Fb1 + Fb2 + ÆÆÆ + Fbn]/[N], where Fb, frequency of the specific behavioStages of lactation: 1st (before challenge), 2nd (D1–D13), 3rd (D14–D27).a,b,c Different letters within the same row define statistically significant differenk,l Different letters in adjoining cells within the same stage define statistically s

* Mean ± standard error of the mean.

3.5. Behavioural findings

3.5.1. Ewe behaviour

No significant changes in specific behaviours wererecorded among group I ewes during the study(P > 0.05). Conversely, there was a progressively increasingfrequency of ‘‘hindering sucking’’ and of ‘‘head up pos-ture’’ among group C ewes subsequent to D0 (P = 0.016and P < 0.001, respectively). It is also noteworthy that,during the third stage of lactation, ‘‘hindering sucking’’usually coincided with the end of ‘‘sucking bouts’’. Withtime, there was also a progressively decreasing frequencyof ‘‘vocalisation’’ subsequent to D0 (P = 0.011).

During the third stage of lactation, there was a signifi-cantly increased frequency of ‘‘hindering sucking’’(P = 0.045) and ‘‘head up posture’’ (P = 0.003) by groupC compared to group I ewes; conversely, there was a signif-icantly increased frequency of ‘‘vocalisation’’ (P = 0.013)by group I compared to group C ewes (Table 3).

3.5.2. Lamb behaviours

There was a progressively decreasing duration of ‘‘suck-ing attempt’’, ‘‘successful suck’’ and ‘‘sucking bout’’ fromthe first to subsequent stages of lactation for group I andgroup C lambs (P < 0.025 and P < 0.045, respectively).There was also a progressively decreasing frequency of‘‘vocalisation’’ (P = 0.040 and P = 0.006, respectively).Finally, a progressively increasing frequency and durationof ‘‘eating’’ behaviour of lambs of both groups(P < 0.001 and P = 0.001, respectively) was found.

culation with S. simulans (group I) or controls (group C) and of their lambs

Stage 3rd Stage

C I C

5 ± 0.08 1.48 ± 0.18 2.39 ± 0.05 2.89 ± 0.154 ± 0.34 2.26 ± 0.53b 3.38 ± 0.29k 5.91 ± 0.36c,l

8 ± 0.11 0.02 ± 0.02 0.17 ± 0.14 0.15 ± 0.192 ± 0.10 0.52 ± 0.26 0.18 ± 0.12 0.20 ± 0.161 ± 0.34 1.58 ± 0.36 2.32 ± 0.29 4.97 ± 0.334 ± 0.19 0.68 ± 0.21a 1.05 ± 0.25k 3.72 ± 0.11b,l

1 ± 0.79k 0.38 ± 0.51b,l 0.38 ± 0.29k 0.20 ± 0.87b,l

0 ± 0.00 0.19 ± 0.19 0.00 ± 0.00 0.00 ± 0.005 ± 0.24 0.02 ± 0.02 0.07 ± 0.11 0.13 ± 0.420 ± 1.01 3.68 ± 0.63 4.79 ± 0.95 9.08 ± 2.02

4 ± 0.09 5.22 ± 0.12 4.95 ± 0.06 5.91 ± 0.093 ± 1.05k 14.34 ± 1.85l 8.73 ± 0.30k 12.13 ± 0.52l

4 ± 1.04k 12.37 ± 1.70l 7.33 ± 0.21k 9.74 ± 0.46l

3 ± 0.16 4.02 ± 0.39 3.71 ± 0.11 4.97 ± 0.190 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b

8 ± 0.29b 2.99 ± 0.64b 4.80 ± 0.24b 5.70 ± 0.43b

ur per animal; N, number of animals in group.

ce (P < 0.05).ignificant difference (P < 0.05).

Table 4Mean frequency (mFb) and mean proportion (%) of duration (mDb) of ‘‘Sucking attempt’’ and ‘‘Successful suck’’ of lambs of ewes challenged byintramammary inoculation with S. simulans (group I) or controls (group C) in relation to the mammary gland sucked by the lamb on each occasion

Behaviour abbreviation 1st Stage 2nd Stage 3rd Stage

Group I

‘‘i’’ ‘‘c’’ ‘‘i’’ ‘‘c’’ ‘‘i’’ ‘‘c’’Sucking attempt (mFb) 4.28 ± 0.84* 6.28 ± 1.12 5.35 ± 1.35k 9.44 ± 0.77l 2.81 ± 0.40k 5.47l ± 0.25l

Sucking attempt (mDb) 1.2 2.7 1.0k 3.0l 0.4k 1.2l

Successful suck (mFb) 3.19 ± 0.88 5.41 ± 1.55 4.35 ± 1.28k 8.31 ± 0.69l 2.30 ± 0.32k 4.64l ± 0.19l

Successful suck (mDb) 1.1 2.6 0.9k 2.9l 0.4k 1.1l

Group C

‘‘p1’’ ‘‘p2’’ ‘‘p1’’ ‘‘p2’’ ‘‘p1’’ ‘‘p2’’Sucking attempt (mFb) 4.36 ± 1.64 8.09 ± 1.34 5.16 ± 2.18 8.26 ± 1.12 4.87 ± 0.61 6.82 ± 0.46Sucking attempt (mDb) 1.2 2.7 1.1 2.9 0.7 1.1Successful suck (mFb) 3.51 ± 1.92 7.30 ± 1.31 4.35 ± 2.03 7.17 ± 1.04 3.95 ± 0.52 5.39 ± 0.40Successful suck (mDb) 1.1 2.6 1.0 2.8 0.7 1.2

mFb = Fb1 + Fb2 + ÆÆÆ + Fbn]/[N], where Fbi, frequency of the specific behaviour per animal; N, number of animals in the group.MDb = [Db1 + Db2 + ÆÆÆ + Dbn]/[N], where Dbi, proportion of duration of the specific behaviour per observation slot per animal (ewe or lamb); N,number of animals in the group.Stages of lactation: 1st (before challenge), 2nd (D1–D13), 3rd (D14–D27).i: Inoculated glands of I ewes; c: Control glands of I ewes.p1: Two right and two left glands, selected at random, of C ewes; p2: The other two right and two left glands of C ewes.k,l Different letters in adjoining cells within the same stage define statistically significant difference (P < 0.05).

* Mean ± standard error of the mean.

0

3

6

9

12

3rd2nd1st

Mea

n fre

quen

cy (m

Fb)

i -SAc-SAi -SSc-SS

382 D.A. Gougoulis et al. / The Veterinary Journal 176 (2008) 378–384

After D0, there was also a significant difference in fre-quency of ‘‘sucking attempt’’ (P = 0.044 for second stage,P = 0.009 for third stage) and ‘‘successful suck’’ (P =0.032 for second stage, P = 0.004 for third stage) behav-iours between group I and group C lambs; however, therewas no significant difference for ‘‘sucking bout’’ (P = 0.283and P = 0.155, respectively) (Table 3).

There was a significantly increased frequency and dura-tion of ‘‘sucking attempt’’ and ‘‘successful suck’’ to controlglands (‘‘c’’), rather than challenged glands (‘‘i’’) by group Ilambs (P < 0.045 for second stage and P < 0.010 for thirdstage); these differences first became evident on D3(P < 0.05) and were maintained until the end of the study.Conversely, no such differences were observed for group Clambs (‘‘p2’’ glands vs ‘‘p1’’ glands) (P > 0.060 for secondstage and P > 0.250 for third stage) (Table 4, Fig. 1).

Before D0 (Stage 1), lambs in group I started the ‘‘suck-ing bouts’’ (n = 130) at equal proportions from ‘‘c’’ (58%)and ‘‘i’’ (42%) glands (P = 0.267). After D0, they startedthe ‘‘sucking bouts’’ (Stage 2: n = 178, Stage 3: n = 96)more frequently from ‘‘c’’ glands (Stage 2: 68%, Stage 3:76%) than from ‘‘i’’ glands (Stage 2: 32%, Stage 3: 24%);these differences were significant (Stage 2: P = 0.001, Stage3: P = 0.05). Conversely, lambs in group C started the‘‘sucking bouts’’ (Stage 1: n = 61, Stage 2: n = 76, Stage 3:n = 64) at equal proportions from ‘‘p1’’ glands (64%,46%, 42%, respectively) and ‘‘p2’’ glands (36%, 54%, 58%,respectively) (P = 0.65, P = 0.643, P = 0.65, respectively).

Stage of lactation

Fig. 1. Mean frequency (mFb) of ‘‘Sucking attempt’’ (SA) and ‘‘Success-ful suck’’ (SS) behaviours of lambs of ewes challenged by intramammaryinoculation with S. simulans in inoculated (i) or control (c) mammaryglands sucked by the lamb on each occasion, during the 1st (beforechallenge), 2nd (D1–D13) and 3rd (D14–D27) stages of lactation.

3.6. Bacteriological and pathological findings

S. simulans was isolated in pure culture from tissue sam-ples collected from 5/8 challenged mammary glands. No

bacteria were isolated from tissue samples collected fromcontrol glands of group I ewes or from any gland of groupC ewes.

In histological sections from all challenged glands, fea-tures of chronic inflammation predominated and inter-alveolar lymphocytes, areas of alveolar destruction andfibrous tissue proliferation between the remaining alveoliwere evident. However, areas with features of acute inflam-mation (neutrophilic infiltration) co-existed. These lesionswere observed in 8/8 challenged glands. All tissue samples

D.A. Gougoulis et al. / The Veterinary Journal 176 (2008) 378–384 383

obtained from control glands (group I or group C ewes)appeared normal.

4. Discussion

Milk yield reduction observed in subclinical mastitis isthe consequence of destruction of glandular elements ofthe mammary gland caused by the invading microorganism(Fthenakis and Jones, 1990b). In the present study, we con-firmed induction and establishment of subclinical mastitisby means of bacteriological, cytological and histopatholo-gical examinations. The results also provided clear evidencethat subclinical mastitis results in changes in patterns ofmaternal–offspring behaviour, which have not been studiedpreviously.

Although pain was not directly assessed, there weresome changes in behaviour that could indicate discomfortin the affected ewes. These included increased ‘‘vocalisa-tion’’ and reduced ‘‘head-up posture’’ behaviours, whichare used by ewes to ‘‘call’’ their lambs to suck (Pickupand Dwyer, 2001, 2002). Surprisingly, ‘‘hindering sucking’’,a behaviour expected to be indicative of ewe discomfort,was actually decreased in challenged ewes. These changesin behaviour indicate impaired ewe welfare due to subclin-ical mastitis.

No preference has been demonstrated among singlelambs for sucking a particular mammary gland of theirdam (Ewbank, 1967; Hess et al., 1974). This was confirmedamong control lambs in the present study, as well as amonglambs of inoculated ewes in the period before challenge.However, these lambs subsequently changed that patternand were found to suck the uninoculated gland of theirdam more frequently. The frequency and duration of suck-ing was largely proportional to milk yield of control andinfected glands.

This change of normal behavioural patterns wasobserved by 3 days post-infection. In subclinical mastitis,reduction of milk yield occurs by 1 day after infection (Fth-enakis and Jones, 1990b; Saratsis et al., 1999). As thechange in behavioural patterns was recorded on D3, onemay postulate that lambs sensed relatively quickly thatthe challenged gland could not fulfill their milk require-ments. It is possible that the salty taste of milk from mas-titic glands (Oliveira et al., 2002) might affect lambpreference. The observed differences in frequency of ‘‘suck-ing attempt’’ and initiation of ‘‘sucking bouts’’ indicatethat lambs appreciated the difference between the twoglands of their dam. Thus, they approached the healthygland more frequently.

However, there was no significant difference in fre-quency and duration of sucking bouts among group Ilambs and group C lambs. Sucking bouts included totalsucking attempts and successes in relation to both glandsof their dam. Therefore, it seems probable that ultimatelythe lambs of the two groups consumed similar quantitiesof milk. This was reflected in the lack of any growth orweight differences between lambs of the two groups.

In dairy ewes, diagnosis of subclinical mastitis can bereadily carried out by bacteriological and cytological exam-ination of the milk. However, we suggest that in meat-typesheep breeds, where milk testing is rarely carried out,altered lamb behaviour could be used as a means of diag-nosing subclinical mastitis.

5. Conclusion

The behavioural implications of subclinical mastitishave not been described previously. In this study, we dem-onstrated that subclinical mastitis results in a reduction inmilk yield in the affected mammary gland and a preferencefor lambs to suck from the unaffected gland. Although noeffects on growth of lambs were observed, their suckingbehaviour was clearly altered. There would be value instudying the behavioural effects of subclinical mastitis inewes rearing twins. These observations can also be usedas a model in cases where ewes in early lactation are poorlyfed and fail to yield sufficient milk. The findings underlinethe need for confirmation of the health status of the mam-mary gland in future studies of lactational behaviour inorder to avoid conflicting results and standardise proce-dures in ethological research.

Acknowledgment

The project was co-funded by the European Social Fundand National Resources – EPEAEK II-PYTHAGORAS.

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