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In recent years programs for the control of mastitis in goats and sheep have been widely applied, resulting in a very important decrease in the prevalence of clinical and subclinical mastitis. This improvement is easily demonstrated by the moving average of the monthly somatic cell count (SCC) of a given farm or cooperative.

In addition to these programs for the control of mastitis, other important changes are observed: increased professionalization of farmers and veterinarians, an increase in the average size of the farms, a tendency towards stabling and the substitution of autochthonous races with increased production livestock, etc.

All these transformations necessarily imply many changes in the handling of the animals that in turn lead to a change in the etiology of the diseases observed. A change in the orientation of our prevention strategies is therefore needed.

The present study describes the evidence of these changes in the etiology of mastitis in small ruminants, with the recommendation of a series of control measures.

MATERIAL AND METHODS

Somatic cell count (SCC)

The SCC of each farm was obtained from the data supplied by the Interprofessional Dairy Laboratory of Castilla and León (LILCyL). An annual average was obtained from each of the farms of two cooperatives. Within each cooperative a mean annual SCC was obtained for the global cooperative by averaging the SC corresponding to each of the farms, regardless of the number of animals on each individual farm.

Samples

In the laboratory (Exopol) samples were obtained from 3351 different farms comprising sheep (2714 herds) and goats (637 herds) and located throughout Spain. The samples corresponded to a period between January 1995 and December 2000, and were distributed by animal species and year, as reflected in Table 1. In each shipment the veterinarian included between one and 20 milk samples (often including a sample from the tank). In all cases the samples corresponded to animals that had yielded positive California tests. For the purpose of the present study, the samples corresponding to clinical mastitis were not differentiated from those corresponding to subclinical disease.

Table 1. Total number of cases of mastitis in goats and sheep recorded each year, and the number of cases in which coagulase-negative staphylococci (CNS), Staphylococcus aureus or Streptococcus agalactiae was isolated in some of the samples, or the presence of Mycoplasma spp. was detected via immunoperoxidase (IPX).

Microbiological culture

Each individualized milk was seeded in blood agar and MacConkey (Oxoid), followed by incubation under anaerobic conditions at 37ºC for 72 hours. The isolated bacteria were identified with the API 20E system (Biomerieux) or via biochemical tests following standard protocols (Carter and Cole, 1990).

All the milk samples received were seeded in Friis and Modified Hayflicks's medium for Mycoplasma, supplemented with thallium acetate 200 µg/ml, penicillin 100 IU/ml, horse serum (10% v/v) and pig serum (10% v/v). In all cases, and to maximize the recovery of Mycoplasma, the samples were preincubated in broth for 3-7 days, followed by incubation in agar for up to 15 days, at 37ºC. The suspect colonies were in turn subcultured and stored frozen.

Detection of Mycoplasma antigens by indirect immunoperoxidase

Of the milk samples received from each farm, a pool was prepared from 6-10 different milks, or alternatively the tank sample was directly used. The cells in milk were obtained by centrifugation in 1.5 ml Eppendorf tubes (1000 g for 5 min.), and were washed three times with PBS.

The separated cell population was then adjusted in each case to a concentration of 5 - 6 x 10⁵ cells/ml in a Newbauer chamber, followed by the deposition of 10 µl of the cell suspension onto multisphere slides. The cells were fixed for 30 min. in 20% acetone solution in PBS 0.01M pH 7.2 containing 0.1% Tween 80, and 0.05% Cas-block, (Zymed, 008020).

The nonspecific inhibitors and endogenous peroxidase were minimized by postfixation for 10 min. in 0.3% hydrogen peroxide. After a series of washings in PBS-Tween, 10 µl of the dilution of primary antibodies per sample were deposited for one hour in a humid chamber at 37 ºC. The negative test control consisted of rabbit serum free of Mycoplasma (Sigma S-3509), or antibody diluent (PBS 0.01 M pH 7.2). The second antibodies conjugated to peroxidase were then applied to the samples and to the controls, based on the same incubation parameters as before.

The resulting immune complexes were in turn developed with 9-amino-3-ethyl carbazole (AEC, Sigma A-6926) in dimethylformamide (DMF, Sigma D-4254) and acetate buffer 50 mM pH 5, applied for 10 min. at room temperature and in the dark. The reaction was stopped with distilled water, followed by contrasting with Mayer’s hematoxylin (Zymed 00-8001). The laminas were mounted with GVA (Zymed 00-8000) and examined under a clear field microscope at x1000 magnification.

The primary antibodies used were: Mycoplasma agalactiae, capricolum, capripneumoniae, mycoides and putrefaciens, and were obtained from the Public Health Laboratory Service. National Collection of Type Cultures. PHSL. 61 Colindale Avenue, London NW9 5HT (England). The second antibody consisted of rabbit monoclonal anti-IgG conjugated with peroxidase (Sigma A-9452, MAb clone GT-34).

In the case of the milk from sheep, only one antibody against M. agalactiae was normally used, while for the samples obtained from goats a pool of the 5 antibodies was employed.

RESULTS AND DISCUSSION

Figure 1 shows the evolution of the mean SC of all the farms belonging to two cooperatives. A clear decreasing trend in SCC is observed, resulting from the implementation of programs for subclinical mastitis control, fundamentally based on revision of the milking machines an routines, teat washing, drying treatments and the use of autovaccines.

Fig. 1. Annual evolution of the mean SCC of the farms belonging to two cooperatives in Castilla and León.

In the laboratory and in some of the milk samples received corresponding to the 3351 cases studied, coagulase-negative Staphylococcus (1663 cases), Staphylococcus aureus (903 cases) and Streptococcus agalactiae were isolated (230 cases), and the presence of Mycoplasma spp. was detected via immunoperoxidase (1212 cases). Other much less frequently isolated microorganisms were: Pasteurella multocida and Mannheimia haemolytica (72 cases), yeasts (36 cases), Aspergillus spp. (33 cases) and Enterobacteria (570 cases).

In nature, the role of each pathogen changes as its environmental conditions modify. For example, parasites are not a major problem on intensive farms where the animals are not put to pasture and have limited contact with their defecations. In the same way, in the context of programs for the control of mastitis, environmental changes have been introduced that have been able to modify the role played by each of the implicated pathogens.

Considering the small number of isolations involving P. multocida, M. haemolytica, yeasts, Aspergillus spp. or Enterobacteria, the variations in their incidences have not been examined in the present work. However, clear tendencies in relation to other pathogens have been observed in both sheep (Fig. 2) and goats (Fig. 3).

Fig. 2. Evolution of the incidence of mastitis due to different pathogens in sheep. Percentage of cases in which the cited pathogen was isolated in some sample.

Fig. 3. Evolution of the incidence of mastitis due to different pathogens in goats. Percentage of cases in which the cited pathogen was isolated in some sample.

Both sheep and goats showed a clear decrease in the frequency of mastitis caused by coagulase-negative Staphylococcus and S. agalactiae, while on the other hand the percentage of cases in which S. aureus was seen to be implicated did not change at all, and the incidence of M. agalactiae in sheep and Mycoplasma spp. in goats actually clearly increased.

The results expressed here cannot be regarded as indicative of the prevalence of the disease, because samplings of the animals and explorations were in no case randomized, and many sources of bias were probably introduced. However, the results do reflect the widespread impression among veterinarians in the field that the etiologies underlying mastitis are evolving.

Streptococcus agalactiae and coagulase-negative Staphylococcus

S. agalactiae is a strictly intramammary pathogen that cannot survive in the environment or in the skin of the animal. Moreover, and unlike in bovine cattle, small ruminants spend periods of time in which no animal is milked. In our opinion, systematic drying treatment has completely eliminated this pathogen from many farms, by arresting its life cycle, since in certain moments all animals on the farm are dry and have been treated in drying — a situation that does not occur in bovine cattle. In fact, our results show a clear tendency towards the eradication of this pathogen in small ruminants.

Coagulase-negative Staphylococcus colonizes the interior and the skin of the udder, and our results in both sheep and goats appear to confirm that mastitis control programs, and fundamentally drying treatment and the washing of teats, suffice to substantially control these pathogens. In fact, both sheep and goats show a marked and continuous decrease in incidence.

Staphylococcus aureus

S. aureus appears much more resistant to the strategies implemented for the control of other staphylococci. In our opinion, although the control of S. aureus essentially requires activities such as the washing of teats, drying treatments or the revision of milking machinery and routines, the capacity of this pathogen to become chronic is the main factor that has prevented a reduction in prevalence.

Once Staphylococcus aureus fixes to a tissue (1) or to the mammary parenchyma of sheep or goats, the formation and growth of a biofilm takes place (2), thus protecting the bacterial microcolony. The latter in turn releases individual bacteria which spread to colonize other zones or animals (3). It is necessary to prevent the formation of such microcolonies in order to avoid the problem of chronic mastitis.

In many cases clinical and gangrenous mastitis due to S. aureus is associated to alterations of the milking machinery, or to skin problems in the sheep (ecthyma, impetigo, etc.). In this sense, it is sufficient to control these problems to maximally limit mastitis. however, minimization of chronic mastitis caused by S. aureus also requires eliminating the animals with chronic lesions, marking and milking at the end those animals with mastitis, and the use of autovaccines specifically targeted against the exopolysaccharides implicated in the formation of microcolonies.

Mycoplasma spp.

In contrast to all the other pathogens studied in Fig.s 1 and 2, a clear increase was observed in the percentage of cases in which the presence of Mycoplasma spp. was detected. Probably, the elimination and control of other pathogens provided an ecological refuge for Mycoplasma. In addition, our mastitis control protocols normally do not contemplate measures to specifically control Mycoplasma transmission, except as regards the control of herd milking routine.

More specifically, to our knowledge none of the commercial drying treatments are active against Mycoplasma, and on the other hand the microorganisms do not colonize the skin of the animals. Sealing is therefore ineffective. In this way Mycoplasma control is almost exclusively limited to the control of milking routines to avoid horizontal transmission.

A program for the control of Mycoplasma should be based on the following points.

- Minimize vertical transmission using artificial colostrum or pasteurized formulas. This limits infection caused by Mycoplasma, Maedi Visna CAE, Coxiella and Paratuberculosis. Artificial lactation is also useful for reducing problems caused by ecthyma, Staphylococcus and Paratuberculosis, because nursing animals become infected by suckling upon udders that have in turn become infected by contaminated feces. On the other hand, caution is required, since bovine colostrum and milk may also carry Mycoplasma, Coxiella and Paratuberculosis. Pasteurization at 65.5ºC for one hour eliminates Mycoplasma and M. paratuberculosis.

- As far as possible, the sharing of pastures and common spaces with other herds should be avoided.

- Maximum hygiene is necessary when milking, in order to avoid horizontal transmission.

- Considering the lack of active intra-mammary treatments against Mycoplasma, many clinicians complete drying with intramuscular treatment using some product active against Mycoplasma. Some veterinarians have even preferred to replace intramammary drying treatment with intramuscular therapy.

- In order to minimize the appearance of Mycoplasma, it is advisable to vaccinate all the animals in all lactations, or once every 6 months. In this context, autovaccines afford the advantage of being prepared with the strains isolated on the actual farm.