Study on the possible survival of Staphylococcus chromogenes through the dry period in dairy ewes

Small Ruminant Research 115 (2013) 124–129

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Study on the possible survival of Staphylococcus chromogenes through the dry period in dairy ewes E. Kiossis a , C.N. Brozos a,∗ , E. Petridou b , A. Zdragas c , Th Papadopoulos c , C. Boscos a a

Clinic of Farm Animals, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greece Faculty of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, Aristotle University of Thessaloniki, Thessaloniki, Greece c Veterinary Research Institute of Thessaloniki, National Agricultural Research Foundation (NAGREF), Thessaloniki, Greece b

a r t i c l e

i n f o

Article history: Received 26 June 2013 Received in revised form 17 September 2013 Accepted 22 September 2013 Available online 30 September 2013 Keywords: Ewes Udder Staphylococcus chromogenes Dry period Mastitis

a b s t r a c t CNS (Coagulase-Negative-Staphylococci) in dairy ewes are the most commonly isolated microorganisms during subclinical mastitis and are related to milk loss. The objective of this study was to investigate whether CNS can survive during the dry period, despite the use of drying-off treatment, and persist during the subsequent lactating period in dairy ewes. 47 primiparus dairy Chios ewes were used. All animals were sampled during the last milking before drying-off and they were then randomly assigned into one of two groups. Ewes of the treated group (n = 21) were administered a dry period intramammary preparation (Nafpenzal® ), and ewes of the control group (n = 26) received no drying-off treatment. Immediately after lambing and once every month until the end of lactation, milk samples were collected for microbiological examination and for the determination of SCC. Daily milk yields were also recorded during the sampling days. Twelve out of the total 47 animals were found to carry the same microorganisms during 2 subsequent milking periods (pre and post partum). Staphylococcus chromogenes was the most commonly identified CNS species (7 animals). All 25 isolated Staphylococcus chromogenes strains were subjected to pulsed field gel electrophoreses in order to identify the presence of the same or different clones of the bacteria. Two clones were identified. Both clones were isolated before dryingoff and re-isolated during the subsequent lactation period (>90% similarity). Apart from the first month of lactation, mean milk yield of treated group was higher than that of the control group, but the difference was significant only after the third month of lactation (P < 0.05). Results from the present study suggest that drying-off treatment is beneficial for milk production, but is not advantageous for the elimination of Staphylococcus chromogenes infections. © 2013 Published by Elsevier B.V.

1. Introduction CNS (Coagulase-Negative-Staphylococci) play an important role in a ewe’s udder health, especially for the establishment of subclinical mastitis (Bergonier et al.,

∗ Corresponding author. Tel.: +30 2310994452; fax: +30 2310994452. E-mail address: [email protected] (C.N. Brozos). 0921-4488/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.smallrumres.2013.09.009

2003; Kiossis et al., 2007; Leitner et al., 2001, 2004). Unlike cows, CNS in sheep are the most commonly isolated microorganisms during mastitis and are related to reduction of milk yield which ranges from 10% to 50% compare to healthy ewes (Bergonier and Berthelot, 2003; Contreras et al., 2007; Watson and Buswell, 1984). The prevalence of intramammary infections with CNS in ewes has been reviewed to range between 25% and 93% (Bergonier and Berthelot, 2003). In sheep, effective udder health

E. Kiossis et al. / Small Ruminant Research 115 (2013) 124–129

management during the end of a lactation period is notable for maintaining mammary health during the subsequent lactating period (Fthenakis et al., 2012). This includes the dry period itself, and the administration of intramammary antibiotic preparations at drying-off (Chaffer et al., 2003; Gonzalo et al., 2004). The cure rate of existing infections after treatment is reported between 65 and 95% (Bergonier and Berthelot, 2003; Chaffer et al., 2003; Gonzalo et al., 2004; Hueston et al., 1989). In dairy heifers, some CNS can survive and persist in the udder for several weeks (Aarestrup and Jensen, 1997). The necessity of treating CNS infections during the milking period is questioned based on the evidence of sufficient self cure rate (Pyörälä and Taponen, 2009; Wilson et al., 1999). The objective of this study was to investigate whether CNS can survive the dry period, despite the use of intramammary preparations, and persist during the subsequent lactating period in dairy ewes. 2. Materials and methods 2.1. Animals and experimental design In the present study 47 primiparus dairy Chios ewes, bred under semi-intensive conditions, were used. The animals were machine milked twice a day and during the commencement of the study they were at the end of their first lactating period. At the start of the drying-off procedure all ewes were clinically examined, with special attention given to their mammary glands. Individual milk yield during the last day of milking was <300 ml. Udder drying-off of all animals took place abruptly. After discarding the first two squirts of secretion milk samples were collected aseptically into sterile containers for microbiological examination. Another sample was then collected for the determination of somatic cell count (SCC) in a 10 ml tube containing 10 mg sodium azide. Samples were kept at 4 ◦ C during transportation to the laboratory and the examinations were performed within 2 h of their collection. SCC determination was performed with Fossomatic® 4000 (A/S N. Foss Electric, Hillerød, Denmark). In all cases, samples were collected from both teats of each ewe. The animals were then randomly assigned into one of two groups. Ewes of the treated group (n = 21) were administered a dry period intramammary preparation (Nafpenzal® , Intervet International BV, Boxmeer, The Netherlands), and ewes of the control group (n = 26) received no antimicrobial agent. Immediately after lambing and once every month until the end of lactation, milk samples were collected for microbiological examination and for the determination of SCC, as previously described. Daily milk yields were also recorded during the sampling days.

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added into a block molds and allowed to solidify at 4 ◦ C for 15 min, 2 plugs made by each strain. Agarose plugs were placed in 3 ml EC-buffer [6 mM Tris HCl, pH 8; 1 M NaCl; 100 mM EDTA, pH 8; 0.5% Brij 58 (Sigma Aldrich, Poole, UK); 0.2% sodium desoxycolate (Difco Laboratories, Detroit, USA); 0.5% sodium N-lauroylsarkosine] and lysed overnight at 37 ◦ C. Subsequently, the plugs were washed 4 times in TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) each one for 15 min with gentle agitation at 54 ◦ C and finally stored in fresh TE-buffer at 4 ◦ C until further analysis. Digestion of DNA was performed with 30 U of the restriction enzyme SmaI (TaKaRa, Kyoto, Japan) for 4 h at 25 ◦ C. Restriction fragments of DNA were separated by PFGE using 1% agarose gels in 0.5X Tris-borate-EDTA buffer with CHEFDR III (Bio-Rad, Hercules, CA, USA). Salmonella serotype Branderup strain H9812 digested with 40 units XbaI (TaKaRa, Kyoto, Japan) was used as size standard. Electrophoresis conditions were 14 ◦ C for 21 h, with pulse time ranging from 5 to 40 s at an angle of 120◦ , the voltage was 6 V/cm. Gels were stained with a solution of ethidium bromide and photographed. The band patterns were visually compared and were classified on the basis of identity of the DNA according to the previously described criteria (Tenover et al., 1995). A database containing all the SmaI PFGE patterns was created by using Bionumerics software (version 6.6 Applied Maths, Sint-Martens-Latem, Belgium) from Applied Maths (Sint-Martens-Latem, Belgium), where band patterns over the multiple gels were normalized and compared. Clustering was performed by using the Dice similarity coefficient and the unweighted pair group method with arithmetic means (UPGMA), with 1% of tolerance and 0.5% optimization. The cluster cutoff was set at 90% and the resulting clusters were designated by capital letters. 2.3. Statistical analysis Differences between groups were tested by using the Student’s t-test. The data related to milk yield and SCC were subjected to one-way analysis of variance (repeated measures ANOVA), in order to establish differences between monthly sampling times; differences among means in this case were tested by Duncan’s test. Normality and homogeneity of variance was tested by applying Kolmogorov–Smirnov and Levene’s test, respectively. Significance was taken at the level of P < 0.05.

3. Results During the course of the experiment, 5 and 10 ewes allocated to the treated group and to the control group, respectively, were removed from the study due to health or general condition problems. Data from these ewes were used until removal of the study. 3.1. Genotypic characterization

2.2. Bacteriological examination 100 ␮l of every sample was spread on sheep blood agar 7% (Liofilchem s.r.l., Italy), MacConkey agar and Manitol dextrose agar (MerckK GaA, Darmstadt, Germany) incubated at 37 ◦ C for 48 h under aerobic conditions, in order the presence of strains of Staphylococcus spp., Streptococcus spp., Trueperella pyogenes, Pasteurella multocida, Mannheimia haemolytica, Pseudomonas aeruginosa, Escherichia coli and/or other members of the Enterobacteriaceae family can be detected. All isolates were stored at −80 ◦ C using 15% Brain Heart Infusion Broth (Liofilchem s.r.l. Bacteriology products, Italy) with glycerol for further molecular investigation. The vast majority of the stains were identified, according to Quinn et al. (1999), as Staphylococcus species, while the biochemical identification was performed using the API ID 32 STAPH (bioM␫rieux, Marcy l’Etoile-France). Pulsed-field gel electrophoresis (PFGE) was carried out as the protocol previously described for DNA macrorestriction from Staphylococcus aureus, with slight modifications (McDougal et al., 2003). Briefly, bacteria were grown overnight in 5 ml Brain Heart Infusion broth at 37 ◦ C, harvested by centrifugation and washed by TE buffer (100 mM Tris-base, 100 mM EDTA, pH 8). 100 ␮l of cell suspension in TE containing 1 ␮g of lysostaphin (Sigma Aldrich, Poole, UK) were mixed with an equal volume of 1.8% low melting point agarose in TE. The cell-agarose suspension was

Twelve out of the total 47 animals were found to carry the same species during 2 subsequent milking periods (pre and post partum). Staphylococcus chromogenes was the most commonly identified CNS species (7 animals) and thus selected for further investigation. In detail, 7 animals were found to carry Staphylococcus chromogenes, 3 Staphylococcus aureus, 1 Staphylococcus warneri and 1 Staphylococcus lentus (Table 1). Of the 7 animals with Staphylococcus chromogenes 6 belonged to the treated group and 1 to the control group. All 25 Staphylococcus chromogenes strains that were isolated in different milking periods were subjected to pulsed field gel electrophoreses in order to identify the presence of the same or different clones of the bacteria. Two clones were identified. Both clones were isolated before drying-off and re-isolated during the subsequent lactation period (clones >90% similarity, Dice coefficient UPGMA). All strains showed similar resistance to the same antibiotics (Fig. 1).

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Table 1 Species of Staphylococcus isolated during the last milking before the dry period and during the subsequent milking period of non-treated and treated with drying-off intramammary antibiotic ewes. Number of halves

Number of animals of treated group

Number of animals of control group

7 1 1 3

11 1 2 4

6 0 1 0

1 1 0 3

12

17

7

5

Number of animals S. chromogenes S. warneri S. lentus S. aureus Total

3.2. Udder health status parameters

throughout the lactation period, but this difference was not significant (P < 0.05).

Isolation of Staphylococcus chromogenes and SCC during the last milking before the dry period and throughout subsequent lactating period from treated and control ewes that found to be infected with Staphylococcus chromogenes during drying-off are presented in Table 2. The mean of SCC in samples of both groups from parturition to the end of lactation is presented in Table 3. Significant (P < 0.05) differences of mean SCC within groups were noticed during the course of the experiment. Average SCC of ewes in treated group was lower compared to that of ewes in the control group

PFGE

3.3. Milk production The mean daily milk production in each group throughout lactation is presented in Table 4. In both groups, peak milk production was reached just after the removal of lambs and remained high for 2 months. It was then declined progressively to the minimum milk production recorded during the seventh month of the lactating period. In all months, mean milk yield of treated group was higher than

PFGE

Cluster

A

Cluster

Β

Animal ID

Halve

Lactating period Treatment

R-type

2298

R

2

N

PenAmpAmc

2298

R

2

N

Pen

0872

R

2

N

PenAmpAmc

2298

R

1

N

PenAmpAmc

3016

L

2

N

PenAmpAmcEr

0886

R

1

C

PenAmpAmcEr

0886

R

2

C

PenAmpAmcEr

3016

L

1

N

PenAmpAmcEr

0872

R

1

N

PenAmpAmc

2611

R

1

N

PenAmpAmc

2611

R

2

N

PenAmpAmc

0886

L

2

C

PenAmpAmcEr

0886

L

1

C

PenAmpAmcEr

0604

L

1

N

PenAmpAmcStr

0604

R

1

N

PenAmpAmcStr

0604

L

2

N

PenAmpAmcStr

0604

R

2

N

PenAmpAmcStr

0604

L

2

N

PenAmpAmcStr

0604

L

2

N

PenAmpAmcStr

1212

L

1

N

PenAmpAmcErStr

1212

L

2

N

PenAmpAmcErStr

2298

L

1

N

PenAmpAmc

1212

R

1

N

PenAmpAmcErStr

1212

R

2

N

PenAmpAmcErStr

2298

L

2

N

PenAmpAmc

2298

L

2

N

PenAmpAmc

2298

L

2

N

PenAmpAmc

Fig. 1. Pulsed-field gel electrophoresis (PFGE) patterns for a selected set of Staphylococcus chromogenes isolates, originating from drying-off (1) and after lambing (2), from two groups (N: Treated with Nafpenzal® , C: control) and different halves (L: left, R: right) of seven sheep. The antimicrobial resistance pattern of each isolate is also displayed (Amp: ampicillin; Amc: amoxicillin, Er: erythromycin; Pen: penicillin; Str: streptomycin). The numbers on the branches indicate the similarity levels and the dashed line indicate the 90% similarity cut-off and the clusters A and B. Animal ID: 4 last digits of the formal animal number according to EU regulations; halve, R: right, L: left; lactating period, 1: dry off, 2: after lambing; treatment, N: treated with Nafpenzal® , C: control, R-Type: antimicrobial resistance pattern.

1352 115 425 1580 129 5779 7973

4635 100 392 3085 11 7034 7804 912 312 945 942 168 792 4749 LR – L LR – LR –

SCC R

ID: identity. G: group, treated (N) and control (C). H+: Staphylococcus chromogenes positive for left (L) or right (R) halve(s). SCC L/R: Somatic cell count left or right halves (×103 ).

SCC L

1510 169 836 420 110 711 8701 RL – L – – – LR

H+ SCC R

8889 147 11 930 120 2630 10,281 1979 54 138 360 174 2753 5953

SCC L H+

RL – – – – – – 565 81 69 713 233 565 7487

SCC R SCC L

2835 132 46 126 89 2835 4997 L – – – – L –

H+ SCC R

3374 98 102 1459 223 907 11,147 913 115 59 106 166 1670 3394

SCC L H+

– – – – – – – 180 33 40 754 50 1671 10,205

SCC R SCC L

121 161 46 39 41 3955 8696 – – – R – – R

H+

385 408 65 2747 250 5012 166

SCC R

N N N C N N N 2298 872 3016 886 2611 604 1212

SCC L

116 88 152 786 92 72 1524 – R – LR R R L

H+

SCC R

1610 976 298 2080 778 4670 2235

1st sampling H+

876 305 1100 930 456 2560 3870

5th sampling 4th sampling 3rd sampling 2nd sampling Lactating period Last milking before the dry period

LR R L LR R LR LR

6th sampling

7th sampling

SCC R SCC L H+

4. Discussion

G

SCC L

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that of the control group, but the difference was significant only after the second month of lactation (P < 0.05). The mean (±S.D.) of the total milk yield of ewes in the treated group (266.68 ± 24.22) was significantly higher than that of ewes in the control group (220.50 ± 11.9) (P < 0.05).

ID

Table 2 Isolation of Staphylococcus chromogenes and SCC during the last milking before the dry period and throughout subsequent lactating period from treated (Nafpenzal® ) and control ewes that found to be infected with Staphylococcus chromogenes during drying-off.

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The use of antibiotic preparations at the end of lactation (drying-off treatment) is considered as an effective tool for the treatment of subclinical mastitis, for the prevention of new intramammary infections during the dry period (Chaffer et al., 2003; Gonzalo et al., 2004), as well as for farms with high individual or bulk-milk SCC (Shwimmer et al., 2008). Although self cure rate of subclinical intramammary infections of sheep is reviewed relatively high (35–67%), remained lower compared to the drying-off treated animals (Bergonier et al., 2003). In dairy cows cure rates seem to be even more contradictory (15–67%) (Taponen et al., 2006; Timms and Schultz, 1987). Persistence of the pre-partum isolated clones of Staphylococcus chromogenes during the subsequent lactating period expresses the ability of the microorganism to survive host defense mechanisms (one case) and antibiotic administration (6 cases). In dairy cows it was found that the udder can provide a sufficient self cure rate in mild intramammary infections caused by Staphylococcus chromogenes. Nevertheless, some research groups found that despite antibiotic administration, CNS can persist throughout the lactating period in the infected udder in 76–85% of the mastitis cases (Taponen et al., 2007; Timms and Schultz, 1987). Aarestrup and Jensen (1997) described the presence of Staphylococcus chromogenes in heifers pre and post partum. The possible re-infection was attributed to the presence of the Staphylococcus chromogenes in the micro flora of teat skin. CNS colonize teat ends and this increases the risk of intramammary infection in dairy cows. Some research groups postulate that teat apex colonization with Staphylococcus chromogenes preparture protects quarters of fresh cows (De Vliegher et al., 2003, De Vliegher et al., 2004). Similar data for dairy sheep was not found in the literature. Poutrel (1984) found that in goats in 60% of the cases where CNS had been isolated before the dry period, CNS were re-isolated during the consecutive lactating period. Nevertheless, in this study no intramammary antimicrobial agents were used during the dry period. Data in Table 2 reveals that in two individuals in which Staphylococcus chromogenes had been isolated before entering the dry period (code number of individuals: 872 and 2611) the same strain was re-isolated during the first sampling after lambing, but it was never re-isolated from then on and until the end of lactation (during the consecutive 6 samplings). In addition, SCC in these animals remained relatively low (>250,000 cells/ml), when widely normal accepted value is considered the 500,000 cells/ml (Bergonier et al., 2003). These findings support the hypothesis of possible self-cure in intramammary infections of Staphylococcus chromogenes in dairy ewes. The rest of the ewes in which Staphylococcus chromogenes had been isolated before entering the dry period, there were

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E. Kiossis et al. / Small Ruminant Research 115 (2013) 124–129

Table 3 Mean of SCC (±S.D.) × 103 (cells/ml) of ewes’ halves treated with drying-off antibiotics (Nafpenzal® ) and control group during lactation. Month

1 2 3 4 5 6 7

Mean of SCC (±S.D.) ×103 (cells/ml)

n (udder halves)

Treated group

Control group

Treated group

Control group

369.62 ± 794.27 a/b, x 933.95 ± 381.23 a, x 829.69 ± 2050.21 a/b, x 989.07 ± 2012.70 a/b, x 1107.13 ± 2350.74 a/b, x 999.12 ± 2145.49 b, x 1189.97 ± 2236.30 a/b, x

1801.77 ± 3104 a/b/c, x 1804.44 ± 3850.84 a, x 1692.19 ± 3020.52 a/c, x 1708.68 ± 2980.21 a/b/c, x 1712.06 ± 2811.19 a/c, x 1943.52 ± 2833.63 b, x 1437.37 ± 2733.24 b/c, x

42 42 42 40 37 33 33

52 52 51 48 46 44 32

Means with different letters (x, y) between groups within a month, differ significantly (P < 0.05); means with different letters (a, b, c) between months within a group, differ significantly (P < 0.05).

Table 4 Mean milk yield kg/day (±S.D.) in ewes treated with drying-off antibiotics (Nafpenzal® ) and control group during lactation. Month

1 2 3 4 5 6 7

Mean milk yield kg/day ± S.D.

n (ewes)

Treated group

Control group

Treated group

Control group

1.897 ± .23 1.928 ± .40 b, x 1.755 ± .45 a, x 1.576 ± .44 c, x 1.022 ± .23 d, x 0.728 ± .09 e, x 0.508 ± .0.8 f, x

1.873 ± .25 1.835 ± .31 a, x 1.405 ± .32 b, y 0.965 ± .28 c, y 0.697 ± .14 d, y 0.552 ± .10 e, y 0.453 ± .06 f, y

21 21 21 20 18 16 16

26 26 26 24 23 22 16

a, x

a, x

Means with different letters (x, y) between groups within a month, differ significantly (P < 0.05); means with different letters (a, b, c) between months within a group, differ significantly (P < 0.05).

fluctuations of SCC and Staphylococcus chromogenes was sporadically re-isolated during the post partum samplings. This pattern of Staphylococcus chromogenes infection is difficult to explain and seems that bacteriological culture is not always the exact depiction of the udder’s health in ewes. The same irregular pattern was noticed in other individuals as well, where Staphylococcus chromogenes was not isolated either pre or post partum (data is not presented). The imminent immunological reaction against intramammary infections in small ruminants is expressed with the parallel raise of SCC even during negative microbiological examinations (Leitner et al., 2011; Winter et al., 2003). Staphylococcus chromogenes displayed resistance against the antibiotics used during the drying-off in the present study (Fig. 1). The selection of the specific preparation was made because it is the only one licensed in the Greek market for use in small ruminants. Despite the fact that the drying-off treatment is proven to be helpful for the improvement of udder health in small ruminants, results of the present study demonstrate that animals infected with Staphylococcus chromogenes were not cured and remained carriers of the microorganism throughout subsequent lactation. The beneficial effect of the dryingoff treatment was indicated from the fact that treated animals had lower SCC (Table 3), although insignificant, and higher milk yield (Table 4) compared to the control animals. The difference in milk yield was significant after the second month of lactation. Nevertheless, the use of the drying-off treatment in dairy sheep seems necessary for the control and prevention of udder infections as well as the improvement of milk production (Chaffer et al., 2003; Gonzalo et al., 2002, 2004; Hueston et al., 1989).

Results from the present study indicate the presence of at least a reservoir for the two strains of Staphylococcus chromogenes isolated in the farm were the study took place. The combination of the drying-off treatment and the application of a program for the control of subclinical mastitis in dairy ewes was suggested as advantageous for maintaining mammary health (Kiossis et al., 2007). It seems possible that apart from pharmaceutical treatment, the control of udder infection involves other factors such as immune response, breed, animal and farm management (Albenzio et al., 2002; Bergonier et al., 2003; Contreras et al., 2007). In conclusion, Staphylococcus chromogenes can potentially survive the dry period, despite the use of the drying-off treatment. Infected animals sustained elevated SCC until the end of the subsequent lactation, while cured animals demonstrated decreased SCC. Despite the beneficial effect of the use of the drying-off treatment for the milk production, it seems that this use is not advantageous for the elimination of certain CNS infections. The development of intramammary preparations especially for dairy ewes could be more effective for the control of udder infections.

Acknowledgements We would like to thank National Agricultural Research Foundation (NAGREF), at Paralimni, Giannitsa, for kindly permitting the use of its animals and establishment. We also thank Intervet Hellas for supplying the intramammary antibiotics.

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