Clinical mastitis in ewes; bacteriology, epidemiology and clinical features

Background Clinical mastitis is an important disease in sheep. The objective of this work was to identify causal bacteria and study certain epidemiological and clinical features of clinical mastitis in ewes kept for meat and wool production. Methods The study included 509 ewes with clinical mastitis from 353 flocks located in 14 of the 19 counties in Norway. Clinical examination and collection of udder secretions were carried out by veterinarians. Pulsed-field gel electrophoresis (PFGE) was performed on 92 Staphylococcus aureus isolates from 64 ewes. Results and conclusion S. aureus was recovered from 65.3% of 547 clinically affected mammary glands, coagulase-negative staphylococci from 2.9%, enterobacteria, mainly Escherichia coli, from 7.3%, Streptococcus spp. from 4.6%, Mannheimia haemolytica from 1.8% and various other bacteria from 4.9%, while no bacteria were cultured from 13.2% of the samples. Forty percent of the ewes with unilateral clinical S. aureus mastitis also had a subclinical S. aureus infection in the other mammary gland. Twenty-four of 28 (86%) pairs of S. aureus isolates obtained from clinically and subclinically affected mammary glands of the same ewe were indistinguishable by PFGE. The number of identical pairs was significantly greater than expected, based on the distribution of different S. aureus types within the flocks. One-third of the cases occurred during the first week after lambing, while a second peak was observed in the third week of lactation. Gangrene was present in 8.8% of the clinically affected glands; S. aureus was recovered from 72.9%, Clostridium perfringens from 6.3% and E. coli from 6.3% of the secretions from such glands. This study shows that S. aureus predominates as a cause of clinical ovine mastitis in Norway, also in very severe cases. Results also indicate that S. aureus is frequently spread between udder halves of infected ewes.


Background
Mastitis is an important disease in sheep. Clinical cases are often severe; systemic signs are present and the condi-tion is obviously painful. Clinically affected glands frequently suffer partial or complete damage and do not resume normal function. Reduced milk yield leads to decreased growth of the lambs [1][2][3]. Additional losses associated with clinical mastitis are costs of treatment and culling of ewes due to permanent udder damage [3][4][5][6][7]. In very severe cases, gangrene may develop in the mammary gland and the ewe may die. Thus, mastitis has a major impact on both economy and animal welfare in sheep production.
Differences in climate, production forms and management practices may give rise to differences both in the epidemiology, bacteriology and clinical manifestations of mastitis. In Norway, sheep are kept exclusively for meat and wool production. They are housed during the winter and early spring, including the lambing season.
The objective of this study was to identify bacteria associated with clinical ovine mastitis in Norway. In addition, certain epidemiological and clinical features of the disease were studied.

Animals and clinical data
Udder secretions were collected and clinical data recorded from 509 ewes with clinical mastitis. The ewes belonged to 353 flocks located in 14 counties in Norway ( Figure 1). The geographical distribution of the cases is shown in Table 1. Clinical mastitis was present in one gland in 471 ewes and in both glands in 38 ewes. The study was carried out in 2002, 2003 and 2004. Only cases that occurred between 1 week prepartum and 8 weeks postpartum were included. In Norway, lambing generally takes place in April and May.

Data and sample collection
Thirty-two veterinary practitioners contributed to the study. When called to a case of clinical mastitis, the veterinarian was to examine the ewe, collect udder secretions and record information regarding the identity, age, date of parturition, the number of lambs and the clinical condition of the ewe on a standardized form. Clinical data included the rectal temperature, an assessment of the severity of systemic signs (graded as none, weak, moderate or severe) and local clinical signs of the affected gland, including whether or not gangrene was present (i.e., cold and blue udder and teat skin).
Prior to treatment, samples were collected aseptically from the clinically affected glands in 10-ml sterile plastic vials by the veterinary practitioner according to the International Dairy Federation's standards [23]. Additionally, samples were taken from the clinically unaffected gland of 252 of the ewes with unilateral clinical mastitis. The samples were sent by mail to the laboratory as soon as possible after sampling, or frozen and stored at -20°C until submission.
If a ewe experienced more than one episode of mastitis during the observation period, only the first episode was included in the study.

Microbiological methods
The samples were examined at the National Veterinary Institute or at the TINE Mastitis Laboratory in Molde, Norway, and bacteria were identified according to the recommendations of the International Dairy Federation [23] with additions. The National Veterinary Institute and the TINE Mastitis Laboratory are both quality assured in accordance with NS-EN ISO/IEC 17025. Briefly, the secretions were brought to room temperature, assessed visually and characterized by appearance before they were mechanically shaken and 10 µl plated on Bacto Blood Agar Base No 2 (Difco Laboratories, Detroit, MI, USA) containing 5% washed bovine erythrocytes and incubated for 48 hours in a 5% CO 2 atmosphere at 37°C. Cultures were read at 24 and 48 hours. If growth was not detected after incubation for 24 hours, the original sample was incubated for 4 hours at 37°C and 50 µl aliquots plated and incubated for 24 hours under aerobic (5% CO 2 atmosphere) and anaerobic conditions. Bacterial species were identified tentatively by their gross colony morphology and by Gram staining, and further confirmatory tests were used as necessary. All suspected staphylococcal colonies were tested using the tube coagulase test (Becton Dickinson Microbiology Systems, Bedford, MA, USA). Coagulase-positive staphylococci were streaked on peptone agar (p-agar) (Difco, Sparks, MD) supplemented with 7 mg/l of acriflavin (Sigma-Aldrich Chemie, Steinheim, Germany) [24], and incubated at 37°C for 24 hours. Bacterial growth in the full length of the streak on p-agar was considered confirmative of S. aureus. Isolates identified as S. aureus were stored at -70°C in Bacto Heart Infusion Broth (Difco) with 15% glycerol. E. coli was identified by the lactose and indole tests, and other enterobacteria were identified to the species or genus level by using a microtube identification system (API 20 E ® ; bioMérieux S.A., Marcy-l'Etoile, France). Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus spp. and Enterococcus spp. were distinguished by the CAMP reaction, the aesculin and inulin tests and by culture on the bromthymolblue lactosesucrose agar. Bacteria within the family Pasteurellaceae were identified to the species level by the CAMP reaction, the indole, mannitol, sorbitol, trehalose, dulcitol, oxidase and beta-galactosidase tests and the haemolysis patterns. Clostridium perfringens was differentiated from other Clostridium spp. by colony morphology, immobility and the presence of a zone of partial haemolysis and a zone of complete haemolysis. Arcanobacterium pyogenes was identified by Gram staining and the presence of pinpoint colonies surrounded by a narrow zone of clear haemolysis at 48 hours. None of the samples were from ewes with arthritis, conjunctivitis or pneumonia; therefore, the mammary secretions were not checked for the presence of mycoplasms.
Total DNA was prepared and pulsed-field gel electrophoresis (PFGE) performed as described previously [25] on 92 S. aureus isolates from 21 flocks in which at least one ewe had bilateral S. aureus intramammary infection (IMI) and where at least two ewes experienced clinical S. aureus mastitis. The band patterns were compared visually. Isolates with indistinguishable patterns were considered identical PFGE types while those with at least one band difference were considered to be different types.

Statistical methods
The chi-square test was used to compare the frequencies of cases within different time intervals in relation to parturition and the relative proportions of clinical S. aureus cases and gangrenous mastitis cases in ewes of different parity and with different number of lambs.
The distribution of pairs of S. aureus PFGE types within flocks (equal vs. unequal) in ewes with bilateral IMI was compared with the corresponding distribution that would be expected if all S. aureus isolates found within each flock were paired randomly. All isolates from the flocks that supplied two or more cases of clinical S. aureus mastitis, of which at least one ewe had bilateral IMI, were included, and Fisher's exact test was used to test the probability of identical distributions of the observed and expected pairs. P < 0.05 was considered statistically significant.

Bacteriological and epidemiological findings
The distribution of bacteria cultured from secretions from the glands with clinical mastitis is shown in Table 2. S. aureus was the predominant pathogen and was found in 65.3% of the samples from affected glands. In the samples from the southern, eastern, western and northern regions, S. aureus was found in 76.0%, 59.0%, 63.9% and 69.5%, respectively.
Information about the date of parturition was received from 318 of the 471 cases of unilateral clinical mastitis. The distribution of the observed clinical mastitis cases in relation to the time of parturition is shown in Figure 2. The relative proportion of cases was greatest during the first week after lambing. Sixty-four (20.1%) of the 318 ewes for which the times of lambing and treatment were recorded, were treated for clinical mastitis during the first two days after parturition. The proportion of cases was significantly greater in the first (P < 0.005) and the third (P < 0.05) week postpartum as compared with the second week postpartum.
The proportion of the clinical S. aureus cases among all the clinical cases did not differ significantly between weeks of lactation, between ewes of different parities or between ewes with different number of lambs (data not shown).
In 180 ewes with unilateral clinical S. aureus mastitis, from which samples from both mammary glands were examined, S. aureus was found in secretions from 72 (40.0%) of the glands without clinical signs. In 72 ewes with unilateral clinical mastitis not caused by S. aureus, a subclinical S. aureus infection was found to be present in 10 (13.9%) of the glands without clinical signs.  (Table 3). Given the PFGE types of all S. aureus isolates within each flock, expected flockspecific distributions of PFGE type pairs based on all possible random pairwise combinations of isolates were arranged. The number of pairs with identical types observed in the flocks was significantly greater than that expected, when assuming a random distribution of isolates (P < 0.0001).

Clinical signs
Of the 471 cases of unilateral clinical mastitis systemic signs were recorded in 325 (  all the clinical cases did not differ significantly between weeks of lactation, between ewes of different parities or between ewes with different number of lambs (data not shown).

Discussion
A random and representative selection of sheep flocks was not deemed feasible for this type of study. However, in order to have a reasonably representative geographical spread of the mastitis cases ewes from 14 of the 19 coun-ties in Norway were included. Thus, the present flocks were housed and pastured under various conditions, and the variations in climatic conditions, flock size and management routines of Norwegian sheep production were reasonably well represented. During the lambing season and subsequent weeks the ewes are paid close attention.
Later, most flocks are moved to pastures in the forests or mountains. For this reason, the study was restricted to cases that occurred between 1 week prepartum and 8 weeks postpartum in order to obtain clinical cases of recent origin.
S. aureus was found in 65% of the samples from clinically affected glands. The dominance of S. aureus as a cause of clinical ovine mastitis has also been shown in regional studies in Norway, where S. aureus was isolated from udder secretions of between 64 and 87% of the ewes [26,27]. In the present study, the largest proportion of S. aureus was found in the southern region (76%). A similar proportion of such cases (75%) was previously observed in a study including cases from one of the municipalities of this region [28]. Studies of clinical mastitis in meat sheep in other countries have found varying, though mostly relatively great, proportions of S. aureus infections (20-60%) [5,[17][18][19][20]22]. In a study of dairy sheep in Jordan, Lafi et al. [13] found that 22% of the clinical mastitis cases were caused by S. aureus.
The main S. aureus reservoirs in sheep are suggested to be infected mammary glands and teat lesions [29]. However, S. aureus can also be cultured from intact teat skin and other body sites [10,30,31]. In dairy flocks, transfer during milking is considered an important mechanism for the spread of this organism [29]. In flocks of meat sheep, transmission of S. aureus between ewes could be a result of the herdsman transmitting S. aureus between ewes during manual udder control, or the udder being exposed to bedding material contaminated from infected ewes [6,7]. Some lambs occasionally suck other ewes than their dam, which might be a mechanism for the spread of S. aureus [32]. In Norway, routine examination of teats and udders are performed after weaning, and ewes with palpable abnormalities or which have experienced clinical mastitis are usually slaughtered before the breeding season. This   [5,18]. Enterobacteria, mainly E. coli, were obtained from 7.3% of the clinically affected udder halves, which is similar to the proportions found in other studies on meat sheep [5,18,22]. However, the number of clinical cases caused by Gram negative bacteria and A. pyogenes may be underestimated because the samples were frozen before bacteriological analysis [34,35].
It is noteworthy that nearly 85% of the ewes exhibited systemic signs and that gangrene was present in as much as 9% of the clinically affected udder halves, clearly showing that ovine clinical mastitis must be considered a serious animal welfare problem. However, mild cases of clinical mastitis are most likely underrepresented in this study. According to Norwegian legislation, antibiotic treatment of animals must be initiated by a veterinarian and, for economical reasons, farmers might be reluctant to call for a veterinary surgeon to treat mild clinical cases.
Most mastitis cases occurred close to lambing. One-third of the ewes developed clinical mastitis during the first week after lambing, and a second peak, although somewhat smaller, was observed in the third week postpartum. This is in accordance with data from the Norwegian Sheep Recording System [36]. Likewise, studies in the UK and Ireland found that cases of acute clinical mastitis occurred most frequently during the first week of lactation, while a second peak occurred between the third and fourth week [5] or the fourth and seventh week [18] after lambing.
Clinical cases around parturition might be newly acquired IMI or aggravations of existing subclinical infections [37]. The proportion of very severe cases, in which gangrene had developed, was not greater among cases occurring close to lambing as compared with those occurring later. The second peak could be explained by increased milk demand from the lambs and the eruption of incisors, which increases the risk of teat lesions. It has been reported that teat lesions frequently are present in ewes with clinical mastitis three to four weeks after lambing [38].

Conclusion
This study shows that S. aureus is the most common cause of clinical mastitis in sheep in Norway and that this organism is frequently spread between glands in infected ewes. Further studies identifying predisposing factors, including reservoirs, transmission routes and factors facilitating S. aureus infection of the ovine mammary gland, are needed in order to improve strategies to reduce the occurrence of mastitis in sheep.